This document provides an overview of hearing impairment and deafness. It discusses: 1. Hearing impairment can be isolated or syndromic, with additional non-auditory symptoms. 2. Deafness is classified based on onset as prelingual or postlingual, depending on whether hearing is lost before or after language acquisition. 3. The degree of hearing loss is assessed using audiometric tests and can be mild, moderate, severe, or profound based on decibel loss thresholds. The document examines different aspects of hearing impairment classification including the source of the primary defect, onset timing, and degree of loss.

1. Aziz El-Amraoui (elaz@pasteur.fr)
Institut Pasteur Tunis, 20 Octobre 2016
Approches post génomiques des troubles neurosensoriels,
de la pathophysiologie aux thérapies
1
DEAFNESS & BLINDNESS
AUDITION & VISION
&&
2

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, sifflets
High pitch
Low pitch
Source: cordes vocales, diapason, cordes de piano ou guitare
3
l’oreille
Le son qui travers l’oreille atteint la cochlée où il est converti en signal électrique par un organe sensoriel
spécialisé, l’organe de Corti.
CCI
CCEs
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

3. 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

4. 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

5. 10 kHz (maxi)
< 4 kHz
20 kHz / 160 kHz
< 1 kHz
Outer hair cells (OHCs):
unique to mammals
OHC
Frequencies (KHz)
cambrien
devonian
* Spécialisation de l’épithélium
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 :
9
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

6. Genetics
Aging Noise
DrugsInfections
MULTIPLE CAUSES OF HEARING IMPAIRMENT
11
GENETIC OF HEARING IMPAIRMENT
! Deafness is the most frequent sensory defect and the
major source
! Severe to profound prelingual deafness affects
to 1000 newborns
affected 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)
12

7. 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$$2015
6"
5"
4"
3"
2"
1
DFNB$genes
1995"1996"1997"1998"1999""2000""2001""2002""2003""2004"2005""2006""2007""2008""2009""2010$$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$$2015
2"
1
YXlinked
1995"1996"1997"1998"1999""2000""2001""2002""2003""2004"2005""2006""2007""2008""2009""2010$$2011$$2012$$2013$$2014$$$2015
2"
1
XXlinked
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"
1
DFNA$genes
Beaucoup restent découvrir:
probablement ceux responsables de
formes rares de surdités.
14

8. 100$gènes$iden?fiés
38"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
CDH23OTOFTMPRSS3
MYO3A 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
COCH
MYH14 WFS1
EYA4
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
15
Strategy$for$diagnosis$&$
gene$discovery$
Most$
common
gene?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
16

9. 17
17
 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!
18

10. 19
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
19
Surdités héréditaires:
un gènes pour quelle(s) fonction(s)?
20

11. Quel$est$l’origine$de$la$surdité$?
L’oreille
cellulessensoriellesauditives
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

12. 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
23
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)
24

13. CLASSEMENT EN « CATEGORIES » DES GENES RESPONSABLES DE SURDITES CHEZ l’HOMME
source: Aziz El-Amraoui, Institut Pasteur
25
CLASSEMENT EN « CATEGORIES » DES GENES RESPONSABLES DE SURDITES CHEZ l’HOMME
26

14. 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 -/-
29
27
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
28

15. 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
29
The hair bundle:
the sound receptive structure
F-actin
actin filaments
stereocilium section
tip-link
Stereocilia
30

16. Genes responsible for Usher I syndrome (USH1)
Myosin VIIa
USH1B
Harmonin
USH1C
SANS (USH1G)
Protocadherin 15
USH1F
Cadherin 23
USH1D
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
32

17. Myo 7a Harmonin Cdh23 Pcdh15 SANS
Myo 7a
Harmonin
Cdh23
Pcdh15
SANS
USH1B USH1C USH1D USH1F USH1G
33
Interactions among USH1 proteins
33
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
interstereocilia
extracellular lateral links
34

18. 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
35
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
36

19. 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
38

20. vision loss
NORMAL RETINITIS PIGMENTOSA
39
❖ 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
40

21. RETINITIS PIGMENTOSA
41
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
42

22. 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

23. 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

24. 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 interconnected
by the USH1 protein network.
USH1 in the photoreceptor calyceal processes
USH1
proteins
calyceal
processes
hair bundle
stereocilia
USH1
proteins
CONCLUSION (1)
Sahly I. et al. 2012
48

25. 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

26. Transforme les informations sonores en impulsions électriques pour le nerf auditif
Prothèse 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
52

27. 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

28. 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
56
Ségment
interne
Ségment
externe
Cil
connecteur
Ségment
interne
Ségment
externe
Cil
connecteur
Contrôle Modèle USH1F
56

29. 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/SANS
Scaffold Ankyrin- and Sam-containing protein)
shRNAs against
USH1G
Subretinal injections in the eye:
58

30. 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

31. 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-FC2200 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%)
62

32. 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
63