approches post génomiques des troubles neurosensoriels, de la pathophysiologie aux thérapies
TRANSCRIPT
Aziz El-Amraoui ([email protected])
Institut Pasteur Tunis, 20 Octobre 2016
Approches post génomiques des troubles neurosensoriels,
de la pathophysiologie aux thérapies
1
DEAFNESS & BLINDNESS
AUDITION & VISION
&&
2
In humans, audition (or action of hearing) has two main functions :- communication & vigilance (or alterness)
AUDITION
3
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
3
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
4
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
5
Fonctionnement$et$tonopie$cochleaire
6
HHMI's 1997 Holiday Lectures on Science, Senses and Sensitivity: Neuronal Alliances for Sight and Sound, James Hudspeth http://www.hhmi.org/biointeractive/cochlea
6
Extrêmement$sensible
Très$grande$sélec?vité
Mais$aussi,$extrêmement$vulnérable
7
! 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
8
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
9
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
10
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
13
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¬&all&prelingual&hearing&loss&is&congenital.&&
4. Audiometric&tests&permit&to&assess&the°ree'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
21
Mécanismes$physiopathologiques$à$l’origine$de$la$surdité:$$du$gène$à$la$fonc?on
Sans"(USH1G)
22
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
30
Genes responsible for Usher I syndrome (USH1)
Myosin VIIaUSH1B
HarmoninUSH1C
SANS (USH1G)
Protocadherin 15USH1F
Cadherin 23USH1D
31
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
34
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
37
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
43
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
44
Ultrastructural analysis of photoreceptor cells
inner"segment
outer"segment
connec8ng"cilium
IS:"inner"segment
OS:"outer"segment
45
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
46
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
47
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
48
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
49
Les$mul?ples$défis$de$la$thérapie$pour$le$syndrome$de$Usher
50
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')
51
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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53
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
53
54
Loss of protocadherin-15 leads to disorganization of the subretinal space
(Collab. M. Perron)C. Schietroma
Ratio OS/ONL
Contrôle Modèle USH1F
54
55
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
55
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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
57
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:
58
Engineering of non-human primate model of USH1 retinal dystrophy
59
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
60
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
61
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 ([email protected])!
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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