Aziz El-Amraoui (elaz@pasteur.fr)

Institut Pasteur Tunis, 20 Octobre 2016

Approches post génomiques des troubles neurosensoriels,

de la pathophysiologie aux thérapies

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DEAFNESS & BLINDNESS

AUDITION & VISION

&&

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In humans, audition (or action of hearing) has two main functions :- communication & vigilance (or alterness)

AUDITION

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Hearing is mainly concerned with two parameters of sound :

frequency (wave/sec = Hertz), which allows differentiation of pitch, "

and pressure level (in Decibel, dB), allowing differentiation of

intensity.

The human ear recognizes frequencies between 20 to 20,000 Hz as sounds

Son grave : ex. moteur de camion, sirène de bateau

Son aigu: ex. chants d'oiseaux, siffletsHigh pitch

Low pitchSource: cordes vocales, diapason, cordes de piano ou guitare

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l’oreilleLe son qui travers l’oreille atteint la cochlée où il est converti en signal électrique par un organe sensoriel

spécialisé, l’organe de Corti.

CCICCEs

L’oreille interne regroupe 2 organes sensoriels distincts :

- le vestibule, organe de l’équilibration

- et la cochlée, organe de l’audition

l’oreille interne

4CCI: cellule ciliée interne (3000 cellules/oreille) CCE: cellule ciliée externe (9000-11000 cellules/oreille)

AUDITION

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basilar"membrane

IHC

OHC

The$secret$lives$of$the$inner$ear$sensory$cells$(Hair$cells)

Type of hair cell Number of HC/cochlea Innervation$

INNER HAIR CELLS (IHCs) 3.000-3.500 30.000 fibres (afferents)$

OUTER HAIR CELLS (OHCs) ~ 9.000-12000 3.000 fibres (efferents)

Touffe ciliaire

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Fonctionnement$et$tonopie$cochleaire

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HHMI's 1997 Holiday Lectures on Science, Senses and Sensitivity: Neuronal Alliances for Sight and Sound, James Hudspeth http://www.hhmi.org/biointeractive/cochlea

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Extrêmement$sensible

Très$grande$sélec?vité

Mais$aussi,$extrêmement$vulnérable

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! Deafness is the most frequent sensory defect and the "major source of communication disorders. "

! Severe to profound prelingual deafness affects 1 out of 700 "to 1000 newborns, and ≈ 1 out of 1000 individuals becomes "affected by this condition before 40 years of age. "

! Over 30% of the population by 60 years of age suffer from hearing loss impeding conversational exchanges.

HEARING IMPAIRMENT

$Les$cellules$ciliées$effectuent$$leur$mitose$terminale$avant$de$se$différencier$:$"$$$$$$$$$$$$$$$$Leur$nombre$est$fixé$très$tôt$dans$le$développement$"(10"semaines"de""gesta8on"chez"l'homme).

Toute perte de cellules ciliées auditives chez les mammifères est irréversible

Naissance

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10 kHz (maxi)

< 4 kHz

20 kHz / 160 kHz

< 1 kHz

Outer hair cells (OHCs): unique to mammals

OHC

Frequencies (KHz)

cambrien

devonian* Spécial isat ion de l ’épithél ium

sensoriel :

- Augmentation de la taille du l’épithélium sensoriel

- Allongement de la membrane basilaire; - Diversification des types cellulaires: cellules sensorielles (CCE/CCI); mais aussi cellules de soutien

* Apparition de l’oreille externe * Modifications de l’oreille moyenne

La nécessité d’analyser les hautes fréquences

s’est accompagné de plusieurs innovations morphologiques :

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Evolution of the hearing organ and epithelium

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10 kHz (maxi)

< 4 kHz

20 kHz / 120 kHz

< 1 kHz

Frequencies (KHz)

cambrien

devonian

Perte progressive de"la régénération"des cellules ciliées

Evolution of the hearing organ and epithelium

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Genetics

Aging Noise

Drugs Infections

MULTIPLE CAUSES OF HEARING IMPAIRMENT

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GENETIC OF HEARING IMPAIRMENT

! Deafness is the most frequent sensory defect and the major source

! Severe to profound prelingual deafness affects to 1000 newbornsaffected by this condition before 40 years of age.

! Over 30% of the population loss impeding conversational exchanges.

1866"Mendel"Lois de "l’hérédité 1994"

DFNB1 & DFNB2"Les deux 1er loci "de surdité isolée AR

1992"DFNA1"1er locus "de surdité"Isolée AD

2001"Génome"humain

Demain…

2010"NGS

The identification of deafness genes (1995) continues to improve with technological progress, the human sequence project, & next generation sequencing (NGS)

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Updates: the Hereditary Hearing loss Homepage

(http://hereditaryhearingloss.org)

Grande hétérogénéité génétique

LOCALISATION$AND$IDENTIFICATION$OF$DEAFNESS$GENES$

DFNA DFNB DFNX DFNY AUNA

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IDENTIFICATION$OF$DEAFNESS$GENES$

1995"1996"1997"1998"1999""2000""2001""2002""2003""2004"2005""2006""2007""2008""2009""2010$$2011$$2012$$2013$$2014$$2015

6"5"4"3"2"1DF

NB$gene

s

1995"1996"1997"1998"1999""2000""2001""2002""2003""2004"2005""2006""2007""2008""2009""2010$$2011$$2012$$2013$$2014$$2015

2"1

Auna

1995"1996"1997"1998"1999""2000""2001""2002""2003""2004"2005""2006""2007""2008""2009""2010$$2011$$2012$$2013$$2014$$2015

2"1

YXlin

ked

1995"1996"1997"1998"1999""2000""2001""2002""2003""2004"2005""2006""2007""2008""2009""2010$$2011$$2012$$2013$$2014$$$2015

2"1

XXlin

ked

1995"1996"1997"1998"1999""2000""2001""2002""2003""2004"2005""2006""2007""2008""2009""2010$$2011$$2012$$2013$$2014$$2015

5"4"3"2"1DF

NA$gene

s

Beaucoup restent découvrir: probablement ceux responsables de

formes rares de surdités. 14

100$gènes$iden?fiés38"AD","57"AR,"4"XL,"1"YL 2"(4)"muta8ons"mitochondriales

SMPX

TMC1

TMIE

SLC26A4

MYO15AMYO7AGJB6

MYO3A CLDN14 TRIOBP GRXCR1 RDX

PCDH15 OTOA TECTA USH1C STRC GIPC3

CDH23OTOFTMPRSS3MYO3A COL11A2BDP1MARVELD2

MYO6 HGFESPNESRRB ILDR1 ADCY1 CIB2

WHRN

LOXHD1

TBC1D24OTOGL

GRXCR2TSPEARCABP2GJB3KARSELMOD3

OTOGPTPRQGPSM2TPRN

PNPT1LHFPL5LRTOMT

SLC26A5PJVK

SYNE4MSRB3

MYO1A

DFNA5CEACAM16

DIAPH1

MIRN96

SIX1 CCDC50P2RX2ACTG1

CRYMSERPINB6

GRHL2MYH9

POU4F3

CLIC5

SLC17A8

COCHMYH14 WFS1EYA4

EPS8 DIAPH3 KCNQ4

TJP2 TNC DIABLO PRPS1 POU3F4

COL4A6 TBL1XR1

Connexins, the building blocks of gap junctions

• Les connexines • 26, 30, 31

Molecular diagnosis of deafness

Useful rule of thumb:

Severity of phenotype

Genotype non-inact/ non-inact

inact/ non-inact

inact/inact

more severe

Test rapide: positif dans au moins 45-50 % des cas de surdités profondes chez l’enfant.

GjP2%Cx26

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Strategy$for$diagnosis$&$gene$discovery$

Most$commongene?c$

muta?ons$ for$hearing$loss

Known$$muta?ons$in$the$tested$

popula?on$

Connexin 26/30 (GJB2/GJB6)

" Validated""at""clinical""gene8cs"labs"

" Gene8c"counseling"" Mechanism

Whole$Exome$Sequencing$(WES)Sequencing all

exons of all genes

Targeted$gene$capture$of$ all$genes$underlying$hearing$impairment$&$candidates$

Tartest of about 115 genes:"Hearing panel

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  3D structure, Pr-Pr interactions, and Protein activity

Valida?on$of$«$variants$»$$pathogenicity

in vitro & ex vivo analyses in vivo analyses

in silico analyses

Plasma&membrane&

DATASETS&FACS)Sorted&Hair&Cells&)&RNASeq!Cochleas!and!utricles!were!dissected!from!mice!expressing!EGFP!under!the!Pou4f3!promoter,!specific!to!hair!cells.!Cells!were!dissociated!and!sorted!by!FACS;!hair!cells!(GFP+)!and!surrounding!cells!(GFPG)!were!separately!collected!for!RNA!extracJon.!PolyGA!mRNA!was!selected,!and!the!3'!end!~500bp!of!each!transcript!was!amplified!and!reversely!transcribed!to!cDNA,!and!the!cDNA!was!sequenced!on!an!Illumina!GAGII!or!HiSeq!2000.!This!was!done!at!five!developmental!stages!(E16,!P0,!P4,!P7,!and!P16),!for!both!cochlea!and!utricle.!The!raw!reads!were!quality!filtered!and!mapped!against!the!NCBI!build!37/mm9!mouse!genome!assembly.!The!read!counts!of!all!RefSeq!genes!were!summarized,!normalized,!and!staJsJcal!tests!of!differenJal!gene!expression!for!each!gene!under!various!comparison!schemes!were!performed!using!the!DESeq!package!in!BioConductor.!Publica;on&to&cite:&!Scheffer!D,!Shen!J,!Mingqian!Huang,!Corey!D,!Chen!ZY.!CellGspecific!gene!expression!in!the!inner!ear!with!FACS!and!RNAGSeq!(in!preparaJon)!Funding:!NIDCD!R01DC002281!to!DPC;!NIDCD!R01DC006908!to!ZYC;!Howard!Hughes!Medical!InsJtute!!Download!XLS!GG!will!be!available!as!soon!as!the!manuscript!is!accepted!for!publicaJon!Cochlear&Progenitor&Cells&)&RNASeq!Mouse!cochlear!progenitor!cells!were!dissected!at!E12GE14!and!transduced!with!a!cGMyc!retrovirus!to!create!clonal!cell!lines!that!were!passaged!up!to!100!Jmes!in!culture.!These!lines!maintained!their!mulJpotent!cell!idenJty,!expressing!early!markers!of!the!cochlear!proneurosensory!region.!In!vitro,!the!cells!divided!in!response!to!bFGF!but!entered!into!quiescence!upon!removal!of!growth!factor!and!express!the!cell!cycle!control!genes!Rb!and!p27.!When!cultured!under!the!appropriate!condiJons,!progenitor!cells!can!differenJate!into!neurons,!hair!cells!and!supporJng!cells.!!Total!RNA!was!extracted!from!progenitor!cells!cultured!in!bFGF,!EGF!or!without!growth!factors.!Ribosomal!RNA!was!removed!and!mRNA!was!reversely!transcribed!to!cDNA!via!random!priming.!Libraries!were!generated!from!the!cDNA!and!sequenced!on!the!Illumina!HiSeq!2000.!Data!analysis!was!performed!as!menJoned!above.!!Publica;on&to&cite:&!Kwan!KY,!Shen!J,!Corey!DP.!MulJpotent!innerGear!progenitor!cells!reveal!a!molecular!switch!from!selfGrenewal!to!differenJaJon.!(in!preparaJon)!Funding:!NIDCD!R01DC002281!to!DPC;!AHRF!grant!to!KYK;!Howard!Hughes!Medical!InsJtute!!

©!2012!The!President!and!Fellows!of!Harvard!University!|!Shared!Harvard!Inner!Ear!Laboratory!Database!

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1.  The&source'of'the'primary'defect'defines&different&deafness&types&&

2.  Hearing&impairment&can&be&either&isolated'or'syndromic'(i.e.,&addi;onal&non>auditory&symptoms&or&abnormali;es&are&present).&&

&&3.  The&onset'of'hearing'impairment:&Prelingual'or'postlingual'deafness,&i.e.&hearing&

impairment&beginning&before&and&aCer&the&onset&of&spoken&language,&respec;vely.&All&congenital'(present&at&birth)&hearing&loss&is&prelingual,&but&not&all&prelingual&hearing&loss&is&congenital.&&

4.  Audiometric&tests&permit&to&assess&the&degree'of'hearing'loss:&&''''''''''Mild'(20>40&dB&HL),&moderate'(40>70&dB&HL),&severe'(70>90&dB&HL),&or&profound'(greater&than&90&dB&HL).&&&The&range'of'sound'frequencies'whose&percep;on&is&affected:&

&Low'(<500&Hz),&middle'(500>2000&Hz)&and&high'(>2000&Hz).&&

CLASSIFICATION OF HEARING IMPAIRMENT

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Surdités héréditaires: un gènes pour quelle(s) fonction(s)?

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Quel$est$l’origine$de$la$surdité$?L’

orei

lle

cellu

les

sens

orie

lles

audi

tives

région sensorielle auditive

structures auditives

Synapse

Touffe ciliaire

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Mécanismes$physiopathologiques$à$l’origine$de$la$surdité:$$du$gène$à$la$fonc?on

Sans"(USH1G)

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Descrip8on"du"processus"pathologique

Difficultés,"voire"impossibilité""d’étudier"directement"l’oreille"humaine:"

ex."imagerie"pas"assez"résolu8ve,"âge"du"don"

d’organe"...etc

# " Modèles"animaux:

La souris, un bon modèle expérimental pour l’étude des maladies humaines:

Computed Tomography (CT) Scan

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La souris, un bon modèle expérimental pour l’étude des maladies humaines

scanning EM (hair bundle)

immunolabeling (hair bundle)

ex vivo electrophysiology

Mechano-electrical transduction

current

patch pipefe

s8mulator

3D structureGenetics

Biochemistry:

Pr-Pr interactions (co-IP,

Pull-down...)

Molecular & morphological analyses

in vivo measurements

DPOAEs (distortion products)

Auditory brainstem response (ABR)

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CLASSEMENT EN « CATEGORIES » DES GENES RESPONSABLES DE SURDITES CHEZ l’HOMME

source: Aziz El-Amraoui, Institut Pasteur

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CLASSEMENT EN « CATEGORIES » DES GENES RESPONSABLES DE SURDITES CHEZ l’HOMME

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Comprendre les mécanismes physiopathologiques à l’origine de la surdité

Hair bundle anomalies in deaf mouse mutants

Myosin 7a -/-

Stereocilin -/-miR-96+/-

Whirlin -/- Myosin 6 -/-Pcdh 15 -/-

N1 -/- N1 -/-

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Example:$Les protéines USHER1 et l’organisation de la touffe ciliaire

First cause of deafness-blindness in humans (1 child on 10 000)

Three clinical subtypes : USH1, USH2 and USH3

Hearing loss & vision loss

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THE$USHER$SYNDROME

Vestibular dysfunctionHearing impairment

USH1 Profound "and congenital

Severe Prepubertal"onset

USH2 Mild to severe "and congenital

absent Postpubertal"onset

USH3 Mild and"progressive

variable variable

retinitis pigmentosa

First cause of deafness-blindness in humans (1 child on 10 000)

Three clinical subtypes : USH1, USH2 and USH3

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The hair bundle: the sound receptive structure

F-actin

actin filaments

stereocilium section

tip-link

Stereocilia

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Genes responsible for Usher I syndrome (USH1)

Myosin VIIaUSH1B

HarmoninUSH1C

SANS (USH1G)

Protocadherin 15USH1F

Cadherin 23USH1D

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Weil et al. 1995 Gibson et al. 1995

Alagramam et al. 2001 Ahmed et al. 2001 Alagramam et al. 2001

Weil et al. 2003 Kikkawa et al. 2003

Bork et al. 2001 Bolz et al. 2001 DiPalma et al. 2001

Verpy et al. 2000 Johnson et al. 2003

USH1G SANS Jackson shaker

USH1F Protocadherin 15 Ames waltzer

USH1B Myosin VIIa Shaker-1

USH1 Harmonin deaf circler

USH1D Cadherin 23 Waltzer

phenotype Human gene Protein Mouse

mutant

Ush1d -/-

Ush1g -/-

Ush1c -/-

Ush1f -/-

Ush1b -/-

wild-type

Usher1

Mutant mice for each of the USHER1 genes are all profoundly deaf

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Myo 7a Harmonin Cdh23 Pcdh15 SANS

Myo 7a

Harmonin

Cdh23

Pcdh15

SANS

USH1B USH1C USH1D USH1F USH1G

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Interactions among USH1 proteins

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All USH1 proteins colocalize at the stereocilia tips in the differentiating hair bundle

USH1 proteins + F-actin

El-Amraoui et al. 1996; Boeda et al. 2002; Michel et al. 2005; El-Amraoui & Petit 2005; Michalski et al. 2007, 2009; Lefèvre et al. 2008; Bahloul et al. 2010; Caberlotto et al. 2011; Pepermans et al. 2014

interstereociliaextracellular lateral links

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Boëda et al. 2002; Michalski et al. 2007; Lefèvre et al. 2008; Bahloul et al. 2010

Wild-type

The USH1C proteins (harmonin) in the myosin VIIa-deficient mutant mice?

Stereocilia

Some USH1 proteins are mislocalized in USH1 mutant hair bundle

USH1B mutant

In the absence of myosin VIIaUSH1C

harmonin (USH1C) in the absence of

Myosin VIIa (USH1B) Cadherin 23(USH1D) Protocadherin 15 (USH1F) SANS (USH1G)

Myosin VIIa and SANS are necessary for the transfer of USH1 proteins into the stereocilia

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Step 1: emergence and kinocilum migration

Kinocilium

Mouse stages

expression des protéines Usher

Apical surface of a hair cell Step 2 & 3: fine orientation / bundle differentiation

USH1 proteins and the organisation of the hair bundle

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Protocadherin-15 &

cadherin-23

USH1F/USH1D heterodimers

harmonin (USH1C)

myosin VIIa (USH1B)

SANS (USH1G)

Les protéines USHER et l’organisation de la touffe ciliaire

expression des protéines Usher

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Protocadherin-15 &

cadherin-23

heterodimers

harmonin (USH1C)

myosin VIIa (USH1B)

SANS (USH1G)

La formation de la touffe ciliaire sans les protéines USHER1

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vision loss

NORMAL RETINITIS PIGMENTOSA

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❖ Light induces series of events that end up in the visual sensory epithelium, the retina, within the sensory photoreceptor cells.

The retina, and the visual sensory cells, rod and cone photoreceptors

inner segment (IS)

outer segment (OS)

The outer segment: the light-sensitive structure of photoreceptor cells

Connecting cilium

inner segment (IS)

outer segment (OS)

cone

How the eye works

The eye

~ 2000 opsin molecules are synthesized per min; they all transit through the connecting cilium (CC) to the outer disks

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RETINITIS PIGMENTOSA

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Human Mouse

Congenital deafness Circling behavior

Retinitis pigmentosa

Congenital deafness Circling behavior

No retinitis pigmentosa

? ?

HYPOTHESIS The retinal dystrophy primary defect likely results from a defective pathway common to all

USH1 genetic forms?

THE USHER SYNDROME

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USH1 proteins in mouse photoreceptor cellsand macaque

A strong USH1 proteins labeling is detected at the junction between the inner and outer segments of macaque photoreceptor cells

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Different expression pattern of USH1 proteins in photoreceptor cells between mouse and macaque ?

+ DAPI

Cadherin-23

USH1 proteins in mouse and macaque photoreceptor cells

+ rhodopsin

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Ultrastructural analysis of photoreceptor cells

inner"segment

outer"segment

connec8ng"cilium

IS:"inner"segment

OS:"outer"segment

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PrimateRodent

inner"segment

outer"segment

connec8ng"cilium

No typical calyceal processes in the mouse

photoreceptor cells The calyceal processes were invariably

observed in photoreceptor cells, from macaque,

human and pig species

The inner and outer segments interface

Sahly et al. 2012

F-actin bundles

of the calyceal processes

surround the

opsin-labelled

photoreceptor outer segment

macaqueratmouse

roots

roots

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USH1 proteins in macaque photoreceptor calyceal processes

All USH1 proteins — myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans — are detected in membrane regions between the outer segment basolateral region

and the calyceal processes

USH1 proteins

+

F-actin

F-actin + tubulin

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USH1 in the auditory hair bundle

Despite processing different sensory signals — mechanical and photonic inputs — hair cells and photoreceptors harbor

microvilli-cilium related structures interconnectedby the USH1 protein network.

USH1 in the photoreceptor calyceal processes

USH1proteins

calycealprocesses

hair bundlestereocilia

USH1proteins

CONCLUSION (1)

Sahly I. et al. 2012

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CONCLUSION (2)

All five USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment, especially in the calyceal processes when present.

A defect in USH1-madiated role in the

calyceal processes would cause the

retinal dystrophy in USH1 patients

Rodent photoreceptors do not to rely on the calyceal processes (or USH1 proteins) for their functioning and mechanical stability.

This may accounts for why mice are

poor models for retinal defects

caused by USH1 proteins.

Sahly I. et al. 2012

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Les$mul?ples$défis$de$la$thérapie$pour$le$syndrome$de$Usher

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Transforme les informations sonores en impulsions électriques pour le nerf auditifProthèse implantable, modèles multi-électrodes implantés dès 1970 chez l ’adulte, "1989 chez l ’enfant en France. "Environ 300 implants/an financés par "le ministère pour les surdités congénitales

Le coût global de l'implant cochléaire est estimé à environ 45 000 €."Il comprend les bilans pré-opératoires, l'opération, l'implant lui même et la prise en charge post opératoire (réglages, rééducation).

Partie externe amovible : Partie interne biocompatible : " microprocesseur-boitier; neurostimulateur " micro sur contour; antenne porte-électrodes

Cochlear implants bypass hair cells to stimulate auditory nerve fibers directly

Cochlear implants (also known as 'Bionic Ears')

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we are not just animals

RETINAL THERAPY

Diagnosis of the Usher syndrome2

Modeling Usher retinal dystrophy1

Two objectives:- Confirm the role of the calyceal processes in USH1 retinal dystrophy

- Develop an appropriate pre-clinical model for USH1 retinal dystrophy

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X tropicalis has two Protocadherin-15, which share homology with mammalian CD1 and CD3 isoform

Morpholino-based approach to knock-down USH1F in X tropicalis

(Collab. M. Perron)C. Schietroma

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Loss of protocadherin-15 leads to disorganization of the subretinal space

(Collab. M. Perron)C. Schietroma

Ratio OS/ONL

Contrôle Modèle USH1F

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In the absence of Usher 1F rod calyceal processes are shorter and sparser, and the basal disks are abnormally large

Rod outer segments display structural anomalies

(Collab. M. Perron, Orsay)C. Schietroma

WT morphant

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Ségment interne

Ségment externe

Cil connecteur

Ségment interne

Ségment externe

Cil connecteur

Contrôle Modèle USH1F

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Fundus examination

Optical Coherence Tomography (OCT)

2. FOLLOW-UP PROCEDURES

Global and multifocal ERG

RISC

1b. siRNA-MEDIATED USH1G & USH1C GENE SILENCING

shRNA

siRNA

mRNA recognition and degradation

1a. SUB-RETINAL INJECTION OF shRNA-EXPRESSING AAVs

3. TERMINAL ANALYSES

Establishment of primate models to develop USH1"gene therapy strategies

(Collaboration with Institut de la Vision/plateforme MirCen (CEA))

4. GENE THERAPY

Once retinal degeneration is present, different strategies will be tested to rescue the phenotype, using siRNA-resistant, homologous human cDNAs.

Adapted from Sahly et al., 2012

Source: webvision.med.utah.edu

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Plasmid Design and In Vitro Validation

Engineering of non-human primate model of USH1 retinal dystrophy

USH1G/SANSScaffold Ankyrin- and Sam-containing protein)

shRNAs against USH1G

Subretinal injections in the eye:

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Engineering of non-human primate model of USH1 retinal dystrophy

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Molecular diagnosis of the Usher syndrome

! Type II (USH2) : 4 loci, 3 genes

! Type III (USH3) : 2 loci, 1 gene

! Type I (USH1) : 7 loci, 5 genes

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Access Array IFC échantillons

réactifs

48x48 = 2304 amplifications simultanées

Tampon de lavage

PCR multiplex et NGS

FC1-CS1

CS2-BC-FC2

Région cible

FC1-CS1

CS2-BC-FC2

FC1-CS1

CS2-BC-FC2 200 bases max

~ 200 bases

Mise en place du diagnostic du syndrome de Usher

Genotype/phenotype correlations (evolution of the visual phenotype)

Crystel Bonnet…-> Christine Petit

""Ce#diagnos+c#(fiable#et#efficace)#guide#la#prise#en#charge#médicale#de#ces#enfants#et#éclaire#le#choix#éduca+f#des#parents;#Priorité#doit#être#donnée#à#ces#enfants#pour#l’implantaAon#cochléaire."

"#Ce#diagnos+c#éclaire#le#conseil#géné+que:#il#permet#d’informer#les#familles#du##risque#de#récurrence#du#syndrome#chez#les#enfants#à#venir;#il#indique#le#risque#des#unions#intrafamiliales."

"#Il#est#indispensable#à##la#mise#en#œuvre#de#futures#thérapie(s)##génique#?

Molecular diagnosis of the Usher syndrome

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Access Array IFC

PCRXmul?plexe,$séquençage$haut$débit$et$SNP$array$:$une$neke$améliora?on$du$diagnos?c$moléculaire$du$syndrome$de$Usher$$

En" u8lisant" ces" approches" combinées" (PCR" mul8plex,"séquençage" haut" débit" et" SNP" array)," nous" avons"iden8fié":"•" des" muta8ons" biallèliques" chez" 91.1%" (329/361)" des"pa8ents"•" une" muta8on" unique" pathogène" chez" 5.8%" (21/361)"des"pa8ents"

•"une"muta8on"unique"fauxmsens"prédite"pathogène"chez"1.4%"(5/361)"des"pa8ents"•"aucune"muta8on"chez"1.7%"(6/361)"des"pa8ents

37%"des"pa8ents"avec"une"muta8on"monoallèlique" sont"porteurs"d’un"grand"réarrangement"sur"l’autre"allèle

$15"délé8ons/duplica8ons"sont"nouvelles

358$muta?ons$différentes$dont$224$$sont$nouvelles$(62.5%)

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Promenade autour de la cochlée (Pujol R., Blatrix S. et Pujol T)

http://www.cochlea.org/

How the Ear Works - Nature's Solutions for Listening

http://www.bcm.edu/oto/research/cochlea/Volta/index.html

The Hereditary Hearing Loss Homepage

http://hereditaryhearingloss.org

Transgenic and Targeted Mutation Database, TBASE

http://tbase.jax.org/

The Hereditary Hearing Impairment in Mice site, HHIM

http://www.jax.org/research/hhim

The Institute of Hearing Research Site

http://www.ihr.mrc.ac.uk/hereditary

AUDITION Fonctionnement & dysfonctionnement

Aziz EL-AMRAOUI (elaz@pasteur.fr)!

Deafness'Varia+on'Database'

h"p://deafnessvaria/ondatabase.com/6

OMIM'2'Online'Mendelian'Inheritance'in'Man'

h"p://www.omim.org/6

The'Jackson'Laboratory'

h"p://www.jax.org/6

MGI'Database'at'JAX'

h"p://www.informa/es.jax.org6

NCBI'2'Na+onal'Center'for'Biotechnology'Informa+on'

h"p://www.ncbi.nlm.nih.gov/6

UCSC'Genome'Browser'

h"p://genome.ucsc.edu/6

Ensembl'Genome'Browser'

h"p://www.ensembl.org/6

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