ESPN Proposal for Front Row - ESPN Front Row - Telling Our ...
ESPN-IPNA Master for Junior Classes Glasgow, September 6th ...ipna-online.org/Media/Junior...
Transcript of ESPN-IPNA Master for Junior Classes Glasgow, September 6th ...ipna-online.org/Media/Junior...
ESPN-IPNA Master for Junior ClassesGlasgow, September 6th 2017
Rémi SALOMON
Hôpital Necker - Enfants Malades, Paris
Renal ciliopathies
An
tero
grad
eIF
T-B
Kin
esi
nII
Re
trograd
eIFT-A
Dyn
ein
2
Basal Body
microtubules
Transition fibers
IFT particle
Kinesin IIDynein 2CiliarycomponentCiliarymembrane
Axo
ne
me
Primary cilium – sensory cilium
Tranzition zone:NPHP
Cell cycle dependant
An
tero
grad
eIF
T-B
Kin
esi
nII
Re
trograd
eIFT-A
Dyn
ein
2
Basal Body
microtubules
Transition fibers
IFT particle
Kinesin IIDynein 2CiliarycomponentCiliarymembrane
Axo
ne
me
Primary cilium – sensory cilium
Tranzition zone:NPHP
Cell cycle dependant
An
tero
grad
eIF
T-B
Kin
esi
nII
Re
trograd
eIFT-A
Dyn
ein
2
Basal Body
microtubules
Transition fibers
IFT particle
Kinesin IIDynein 2CiliarycomponentCiliarymembrane
Axo
ne
me
Primary cilium – sensory cilium
Tranzition zone:NPHP
Cell cycle dependant
Tubular cells have a unique cilia at their apical pole
Art and Knoers, Ped Nephrol 2013
Axoneme
Transition zone
Anterograde
transport
Retrograde
transport
Basal body
Motile cilia (9+2)Cellular movement
Flux generation
Nodal ciliaLateralization
Primary cilia (9+0)Ubiquitous
Diverse functionsSignal transduction
9+2 motile cilium 9+2 motile cilium
Oviduct Brain ventricles
Microtubules doublets
Flagellum
Chlamydomonas reinhardtii
Motile cilia (9+2)
Non motile clia (9+0)
Human ciliated cells
Fliegauf 2007
3
9+0 9+2
Primary Ciliary Dyskinesia (PCD)respiratory infection-otitis-situs inversus
Ciliopathiesretina-kidney-liver-CNS-skeleton-situs inversus
node
Hamada, 2002
Connecting ciliumKidney tubesFibroblasts Sperm cellsbronchus
non motile (most) motile (most)
CILIA
3
9+0 9+2
Primary Ciliary Dyskinesia (PCD)respiratory infection-otitis-situs inversus
Ciliopathiesretina-kidney-liver-CNS-skeleton-situs inversus
node
Hamada, 2002
Connecting ciliumKidney tubesFibroblasts Sperm cellsbronchus
non motile (most) motile (most)
CILIA
3
9+0 9+2
Primary Ciliary Dyskinesia (PCD)respiratory infection-otitis-situs inversus
Ciliopathiesretina-kidney-liver-CNS-skeleton-situs inversus
node
Hamada, 2002
Connecting ciliumKidney tubesFibroblasts Sperm cellsbronchus
non motile (most) motile (most)
CILIA
Motile cilia (9+2)Cellular movement
Flux generation
Nodal ciliaLateralization
Primary cilia (9+0)Ubiquitous
Diverse functionsSignal transduction
9+2 motile cilium 9+2 motile cilium
Oviduct Brain ventricles
Nodal flow: the first recognizable LR asymmetry
Extra-embryonic fluid
Nodal flow
Primary cilia : signals downstream
Hedgehog Wnt
(canonical / non canonical PCP)
Chemosensation/receptor
and others ….
Fliegauf 2007
Ciliopathies
• 1000 proteins for cilia generation and maintenance
• More than 100 loci for more than 20 clinical entities
• And probably more than 100 rare human diseases
• The frequency in total might be 1/1000 ( ≈ Down sd)
IFT-188 Tg737
Pazour J Cell Science 2000
Intraflagellar transport
(IFT)
Renal ciliopathies
IFT88: subunit of the IFTB complexidentified in the Chlamydomonas necessary for formation of the flagella
Tg737 mouse: polycystic kidneymouse model. Mutation in the gene encoding IFT88
Mouse
Tg737 mouse: polycystic kidneymouse model (ORPK mouse). Mutation in the gene encoding IFT88
Cystic kidneys
Renal ciliopathies
IFT88: subunit of the IFTB complexidentified in the Chlamydomonas necessary for formation of the flagella
Tg737 mouse: polycystic kidneymouse model (ORPK mouse). Mutation in the gene encoding IFT88
Mouse
PKD is a ciliopathy !
The success of comparative genomics…
Tb: Trypanosoma brucei
Cr: Chlamydomonas reinhardtii
At: Arabidopsis thaliana
Pf: Plasmodium falciparum
Dd: Dictyostelium discoidum
Sc: Saccharomyces cerivisiae
Ce: Caenorhabditis elegans
Hs: Homo sapiens
Dm: Drosophila melanogaster
Li 2004Flagellar Apparatus Basal Body (FABB)
Primary cilia of renal tubules
Sensor of extracellular cues-Growth factors-Morphogens-Light-Fluid flow….
Primary cilium-sensory organelle
Connecting cilium
ChemicosensorMechanosensor
Signal transduction platform
Cell responses
cell proliferationcell differentiationCell migration
Nauli, 2003
Apico-basal polarity
Polar polarity
Polar polarity
Disorientation of the stereocilia in the
inner ear (hair cell of the organ of
corti)
Drosophila mutant (wing)
Planar cell polarity is critical for formation of the renal tubule
• Orientated cell division
→ cystogenesisWnt
(canonical / non canonical PCP)
THORACIC DEFECTS
HEPATIC FIBROSIS
SitusInversus
POLYDACTYLY
STERILITY
CARDIAC DEFECTS
RETINAL DEGENERATION
Adapted from Sarah C. Goetz & Kathryn V. Anderson, 2010
NPH PKD MCKD
Ciliopathies
CEREBELLAR VERMIS HYPOPLASIA
RENAL DEFECTS
Primary cilia of renal tubules
• maladies génétiquescomplexes affectant demultiples organes dûes àdes défauts de formationou fonction des cils
Complex genetic diseases
that affect multiple organs
and that are related to
defect in the formation or
function of cilia
Nephronophthisis
Tubulo-interstitial chronic nephropathy
Autosomal recessive heredity
Polyuria / polydipsia
ESRD around 10-20 yrs (juvenile form))
Histology
Thickening of the basal membrane
Interstital fibrosis(phtisis = shrunken)
Extra-renal anomalies
Genetic heterogeneity +++
Cysts at the cortico-medullar border
• Juvenile NPH
Nephronophthisis
Tubulo-interstitial chronic nephropathy
Autosomal recessive heredity
Polyuria / polydipsia
ESRD around 10-20 yrs (juvenile form))
Histology
Thickening of the basal membrane
Interstital fibrosis
Extra-renal anomalies
Genetic heterogeneity +++
• NPH Infantile
- Cortical cysts (widespread)
- NPHP2/INV and NPHP3
Chrom. 2
NPHP1
NPHP1 gene deletion
PCR
Help
for
diagnosis
NPHP145kb 45kb
~300kb
(1993)
Nephrocystin
(1997)
290 kb
Infantile nephronophthisis mutations in NPHP2 and NPHP3 genes
0 10 20 30 40 50 60 700.0
0.5
1.0
1.5
NPHP2
NPHP3
pas de mutation
Age en mois
pro
po
rtio
n d
e r
ein
ssu
rviv
an
ts
Renal surv
ival
Age (month)
no mutation
An
tero
grad
eIF
T-B
Kin
esi
nII
Re
trograd
eIFT-A
Dyn
ein
2
Basal Body
microtubules
Transition fibers
IFT particle
Kinesin IIDynein 2CiliarycomponentCiliarymembrane
Axo
ne
me
Primary cilium – sensory cilium
Tranzition zone:NPHP
Cell cycle dependant
An
tero
grad
eIF
T-B
Kin
esi
nII
Re
trograd
eIFT-A
Dyn
ein
2
Basal Body
microtubules
Transition fibers
IFT particle
Kinesin IIDynein 2CiliarycomponentCiliarymembrane
Axo
ne
me
Primary cilium – sensory cilium
Tranzition zone:NPHP
Cell cycle dependant
An
tero
grad
eIF
T-B
Kin
esi
nII
Re
trograd
eIFT-A
Dyn
ein
2
Basal Body
microtubules
Transition fibers
IFT particle
Kinesin IIDynein 2CiliarycomponentCiliarymembrane
Axo
ne
me
Primary cilium – sensory cilium
Tranzition zone:NPHP
Cell cycle dependant
Tubular cells have a unique cilia at their apical pole
Cilia not known before 2000
Cilia not known before 2000
Tight junction
Focal adhesion
Nephrocystins not only the cilia N
ep
hro
cys
tin
4/
a-t
ub
ulin
Nep
hro
cys
tin
4/
b-c
ate
nin
MDCK cells
Primary cilia
Cellular junctions
Delay in tight junction formation
Abnormal 3D structures
MDCK knock-down cells
Cystic kidneys/tubulointerstial nephropathy
√ √ √ √ √ √ √ √ √ √ √
Retinal degeneration √ √ √ √ √ √ √ √
Liver defects √ √ √ √ √ √ √ √ √ √
Laterality defects √ √ √ √ √ √
Polydactyly √ √ √ √ √ √ √
Obesity, hypogonadism √ √
Craniofacial anomalies (palate cleft)
√ √ √ √
Cerebellar vermis dysgenesis √ √ √ √ √ √
Neuronal tube defects(encephalocele)
√ √
Skeletal dysplasia (Shortening/bowing of bones )
√ √ √ √
Ectodermal dysplasia √ √ √
i
i
i
OFD: Oro-Facio-Digital
JATD: Jeune Asphyxiating Thoracic Dystrophy
SRP: Short Rib Polydactyly
Normal CHF
Congenital Hepatic Fibrosis (CHF)
defect in the formation of the ductal plate
Aspect of
proliferation of the
biliary canaliculesPortal triad
A
V
CB
Portal triad (liver)
From Gunay-Aygun
AmJMedGenet 2009
Defective remodeling of the ductal plate
Caroli Disease (CD)
Dilations of the medium and large sized intrahepatic bile ducts
Caroli syndrome = CD + CHF
ARPKD ADPKD
From Gunay-Aygun, AmJ Med Genet 2009
Polycystic kidney diseases
Cysts not in continuity
with the intrahepatic
biliary tree
CD
+
CHF
Ciliopathies with Congenital Hepatic Fibrosis/Caroli’s syndrome
From Gunay-Aygun, Am J Med Genet 2009
Liver diseases in ciliopathies
• CHF > portal hypertension
Esophageal varices, hypersplenism
• Caroli disease
Cholangitis
In the long term: cholangiocarcinoma
Liver diseases in ciliopathies
• CHF > portal hypertension
Esophageal varices, hypersplenism
• Caroli disease
Cholangitis
In the long term: cholangiocarcinoma
Cystic kidneys/tubulointerstial nephropathy
√ √ √ √ √ √ √ √ √ √ √
Retinal degeneration √ √ √ √ √ √ √ √
Liver defects √ √ √ √ √ √ √ √ √ √
Laterality defects √ √ √ √ √ √
Polydactyly √ √ √ √ √ √ √
Obesity, hypogonadism √ √
Craniofacial anomalies (palate cleft)
√ √ √ √
Cerebellar vermis dysgenesis √ √ √ √ √ √
Neuronal tube defects(encephalocele)
√ √
Skeletal dysplasia (Shortening/bowing of bones )
√ √ √ √
Ectodermal dysplasia √ √ √
i
OFD: Oro-Facio-Digital
JATD: Jeune Asphyxiating Thoracic Dystrophy
SRP: Short Rib Polydactyly
Hypotonia
Mental retardation
Abnormal breathing pattern
Abnormal eye movements
Joubert syndrome
40 patients
5 isolated and moderate vermis atrophy
2 molar tooth + 9 superior vermis dysplasia
NPHP1
NPHP2• 5 patients
• 5 pericebellar effusion
• 3 vermis atrophy
Cystic kidneys/tubulointerstial nephropathy
√ √ √ √ √ √ √ √ √ √ √
Retinal degeneration √ √ √ √ √ √ √ √
Liver defects √ √ √ √ √ √ √ √ √ √
Laterality defects √ √ √ √ √ √
Polydactyly √ √ √ √ √ √ √
Obesity, hypogonadism √ √
Craniofacial anomalies (palate cleft)
√ √ √ √
Cerebellar vermis dysgenesis √ √ √ √ √ √
Neuronal tube defects(encephalocele)
√ √
Skeletal dysplasia (Shortening/bowing of bones )
√ √ √ √
Ectodermal dysplasia √ √ √
i
i
OFD: Oro-Facio-Digital
JATD: Jeune Asphyxiating Thoracic Dystrophy
SRP: Short Rib Polydactyly
Photoreceptor: the conncting cilia
Rod receptor cell
pigment
epithelium
outer segments
inner segments
outer nuclear
layer (ONL)
outer plexiform
layer (OPL)
inner nuclear
layer (INL)
inner plexiform
layer (IPL)
ganglion cell
layer (GCL)
optic fiber layer
Photoreceptor
Sensory Cilium
Outer segment
Nucleus
Rootlet
Basal body
Transition
Zone
Axoneme
Cell Body
Inner segment
From Rozenbaum, 2002
Connecting cilia
Retinal dystrophy
• Retinal pigmentosa, Leber amaurosis
– Severe > precocious blindness (Senior-Løken)
– Moderate or asymptomatic
• Anomalies of fundus examination
• Anomalies of the electroretinogram
• 10 à 15 % of nephronophthisis cases
Cystic kidneys/tubulointerstial nephropathy
√ √ √ √ √ √ √ √ √ √ √
Retinal degeneration √ √ √ √ √ √ √ √
Liver defects √ √ √ √ √ √ √ √ √ √
Laterality defects √ √ √ √ √ √
Polydactyly √ √ √ √ √ √ √
Obesity, hypogonadism √ √
Craniofacial anomalies (palate cleft)
√ √ √ √
Cerebellar vermis dysgenesis √ √ √ √ √ √
Neuronal tube defects(encephalocele)
√ √
Skeletal dysplasia (Shortening/bowing of bones )
√ √ √ √
Ectodermal dysplasia √ √ √
i
i
i
i
OFD: Oro-Facio-Digital
JATD: Jeune Asphyxiating Thoracic Dystrophy
SRP: Short Rib Polydactyly
Cone-shaped epiphisis>
Saldino-Mainzer sd (eye, liver, cerebellum)
Narrow chest, short ribs>
Jeune sd (asphyxiating thoracic dystrophy)
(eye, liver, retina…)
Ellis Van Creveld sd (heart)
Ectodermic dysplasia >
Sensebrenner sd
Bone ciliopathies
Olfaction
9/19 patients with BBS have anosmia …
Kulaga Nat Genet 2004
Extrarenalassociations (45%):
Eye: retinaldegeneration
(Senior-Løkensyndrome)
Brain: cerebellarvermis
hypoplasia, mental
retardation
(Joubert syndrome)
Liver: fibrosis
Bone: Mainzer-Saldino,
Jeune and Sensenbrenner
syndromes
Heart: ventricularseptal
defect
• SitusInversus
0
5
10
15
20
25
30
35
40
Without known mutation
With identified mutation
NPH Cohort-Necker >900 families
Nephronophthisis : extrarenal features
Renal CiliopathiesDisease Gene Renal disease Extrarenal disease Localization/fun
ction
ADPKD PKD1 Cystic kidneys Liver and pancreatic cysts, intracranial aneurysms, colonic diverticulosis, HTA
BB/cilium
PKD2 Cilium
ARPKD PKHD1 Cystic dilatations Liver, CHF, HTA BB/cilium
Nephronophthisis NPHP1 Juvenile NPH Mild JBTS, RP, Cogan Transition zone
NPHP2/INVS Infantile NPH RP, liver fibrosis, HTA, situs inversus TZ
NPHP3 Juvenile/adolescent/infantile NPH Liver fibrosis, RP, situs inversus TZ
NPHP4 Juvenile NPH Cogan, RP TZ
NPHP5 Juvenile NPH Severe RP TZ, BB
NPHP6/CEP290 Juvenile NPH JBTS, RP TZ, BB, centrosome
NPHP7/GLIS2 Juvenile NPH
NPHP8/RPGRIP1L Juvenile NPH JBTS TZ
NPHP9/NEK8 Juvenile NPH TZ
NPHP10/SDCCAG8 Juvenile NPH RP TZ
NPHP11/MKS3/TMEM67 Juvenile NPH Liver firbosis, CNS, polydactyly, CHF, SI TZ
TTC21B Juvenile NPH osteochondrodysplasia Cilium
AHI1 Juvenile NPH JBTS
Meckel syndrome MKS1/BBS13 Cystic kidney diseaee Liver firbosis, CNS, polydactyly, CHF, SI Cilium/BB
MKS2/TMEM216 Cystic kidney diseaee Liver firbosis, CNS, polydactyly, CHF, SI
MKS3/TMEM67 Cystic kidney diseaee Liver firbosis, CNS, polydactyly, CHF, Si
MKS4/CEP290 Cystic kidney diseaee Liver firbosis, CNS, polydactyly, CHF, SI
MKS5/RPGRIP1L Cystic kidney diseaee Liver firbosis, CNS, polydactyly, CHF, SI
MKS6/CC2D2A Cystic kidney diseaee Liver firbosis, CNS, polydactyly, CHF, SI
Bardet-Biedl syndrome BBS1-BBS13 Various renal phenotypes Obesity, RP, MR, hypogonadism, Basal body
Jeune syndrome IFT80 Various renal phenotypes osteochondrodysplasia, liver fibrosis, PD
DYNC2H1 osteochondrodysplasia, liver fibrosis, PD
Sensenbrenner syndrome IFT122 Various renal phenotypes osteochondrodysplasia,craniosynostosis,
ectodermal anomalies, PDcilium
IFT43 Various renal phenotypes cilium
Orofaciodigital syndrome OFD1 Cystic kidney (PKD like) Oral, facial, digital malformations BB
NPHP3CEP290/NPHP6
RPGRIP1L/NPHP8MKS3/NPHP11
Bergmann C, et al. Am J Hum Genet 2008Balaa, et al. Am J Hum Genet 2007Delous et al., Nature Genetics 2007Otto EA, et al., J Med Genet 2009
High variations for mutation in the same gene
liverkidney
Nephronophthisis Senior-Loken syndrome
Joubert syndrome Meckel syndrome
NPHP2TTC21B
NPHP1
NPHP4
A u g m e n ta t i o n o f t h e s e v e r i t y o f t h e p h e n o t y p e
NPHP5NPHP10
Degeneration Dysplasia
Occipital encephalocelePolydactylyCystic kidney dysplasiaLiver fibrosis
Phenotype/Genotype correlation MKS3/NPHP11
Loss-of-functionon the 2 allelles
Loss-of-function+ hypomorphic
mutation
Hypomorphicmutation onf the
two alleles
Phenotype / Genotype: NPHP6
Leber SLS Joubert Meckel
NPHP6
(CEP290)intronic
missense/splice
truncating truncating
The AHI1 gene p.R830W SNP may increases the phenotype of patients with NPHP1 deletion
Patients with NPHP1 délétion de
(aleles) Controles
(aleles)Neurologic symptoms No neurologic symptoms
5/26 3/152 4/276
p<0.002p<0.001
Tory et al. JASN, 2007
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
TAAATG
R830W
CC WD40
SH3
Patients with homozygous NPHP1 deletion
NPH isolée
NPH + œil
NPH + CNSNPHP1
homozygous deletion
NPHP1 homozygous
deletion + variant
R830W AHI1
NPHP1 homozygous
deletion + variant
R830W AHI1Tory et al., 2007; Louie et al., 2010
Oligogenism and ciliopathies
80%
11%9%RP
CNS
isolatedNPH
Motile cilia
Primary ciliaNPH1848
Oligogenism in complex ciliopathies?
61 E. EscudierCiliated cells from nasal brushing
control NPH1848
Mainzer-Saldino Syndrome
-infantile NPH (ESRD 7 months)
-cone shaped epiphyses
-congenital Leber’s amaurosis
-liver and pancreatic cysts
Bronchial ciliary dyskinesia ???
- Ciliary dyskinesia
- Viral bronchiolitis
- Mental disability – Dilated brain ventricles
Motile cilia
Primary ciliaNPH1848
Oligogenism in complex ciliopathies?
Ciliary
dyskinesia ?
Bi-allelic variations in two genes
GeneNucleotide
Alteration
Protein
Change
Exon (zygosity,
segregation) Polyphen/SIFT
WDR19/IFT14
4
c.3533G>A p.Arg1178Glu 32 (Het, f) 0,948/0,24
del. ex 1-4 - (Het, f) -
TEKT1
c.730C>T p.Arg244* 6 (Het, m) -
c.933G>T p.Lys311Gln 7 (Het, f) 0,286/0,15
Saldino
Mainzer sd
Mainzer-Saldino Syndrome
-infantile NPH (ESRD 7 months)
-cone shaped epiphyses
-congenital Leber’s amaurosis
-liver and pancreatic cysts
Bronchial ciliary dyskinesia ???
- Ciliary dyskinesia
- Viral bronchiolitis
- Mental disability – Dilated brain ventricles
Mainzer-Saldino Syndrome
-infantile NPH (ESRD 7 months)
-cone shaped epiphyses
-congenital Leber’s amaurosis
-liver and pancreatic cysts
Bronchial ciliary dyskinesia ???
- Ciliary dyskinesia
- Viral bronchiolitis
- Mental disability – Dilated brain ventricles
Motile cilia
Primary ciliaNPH1848
Oligogenism in complex ciliopathies?
Additive/synergic effects?
NATURE GENETICS VOLUME 43 | NUMBER 3 | MARCH 2011 189
ARTI CL ES
Genetic and functional studies have shown that defects in genes
encoding components of the ciliary apparatus lead to an overlap-
ping set of clinical phenotypes that include retinal degeneration,
renal cystic disease, polydactyly and other skeletal abnormalities,
fibrosis of various organs, and a complex range of anatomical and
functional defects of the central and peripheral nervous system. The
recognition of the clinical overlap between discrete clinical entities
attributable to ciliary dysfunction has led to the unification of such
disorders under the ciliopathy module1,2. This integration has also
been reflected in the genetic architecture of ciliopathies: although
TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum
Erica E Davis1,2, Qi Zhang3, Qin Liu3, Bi l l H Diplas1, Lisa M Davey1, Jane Hartley4, Corinne Stoetzel5,
Katarzyna Szymanska6, Gokul Ramaswami7, Clare V Logan6, Donna M Muzny8, Alice C Young9,
David A Wheeler8, Pedro Cruz9, Margaret Morgan8, Lora R Lewis8, Praveen Cherukuri9, Baishali Maskeri9,
Nancy F Hansen9, James C Mullikin9, Robert W Blakesley9, Gerard G Bouffard9, NISC Comparative
Sequencing Program9, Gabor Gyapay10, Susanne Rieger11, Burkhard Tönshoff11, I lse Kern12,
Neveen A Soliman13, Thomas J Neuhaus14, Kathryn J Swoboda15,16, Hulya Kayseri li17, Tomas E Gallagher18,
Richard A Lewis19–22, Carsten Bergmann23,24, Edgar A Otto7, Sophie Saunier25, Peter J Scambler26,
Phi lip L Beales26, Joseph G Gleeson27, Eamonn R Maher4, Tania Attié-Bitach28, Hélène Dollfus5,
Colin A Johnson6, Eric D Green9, Richard A Gibbs8, Fr iedhelm Hi ldebrandt7,29, Eric A Pierce3 &
Nicholas Katsanis1,2,30
Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is
underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here
we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated
nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a
large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro
evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes
pathogenic alleles to ~5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with
diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by
functional analysis of all variants informs the genetic architecture of inherited disorders.
1Center for Human Disease Modeling, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA. 2Department of Pediatrics,
Duke University Medical Center, Durham, North Carolina, USA. 3F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania, USA. 4Department of Medical and Molecular Genetics, Institute of Biomedical Research, University of Birmingham, Birmingham, UK. 5Laboratoire de Génétique Médicale EA3949, Avenir INSERM, Université de Strasbourg, Strasbourg, France. 6Section of Ophthalmology and Neurosciences, Leeds
Institute of Molecular Medicine, St. James’s University Hospital, Leeds, UK. 7Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA. 8Human
Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA. 9National Institutes of Health Intramural Sequencing Center, National Human Genome
Research Institute, National Institutes of Health, Bethesda, Maryland, USA. 10Genoscope Centre National de Séquençage, Crémieux, Evry, France. 11University
Children’s Hospital, Heidelberg, Germany. 12Department of Pediatrics, University Hospital of Geneva, Switzerland. 13Department of Pediatrics, Kasralainy School of
Medicine, Cairo University, Cairo, Egypt. 14Division of Nephrology, University Children’s Hospital Zurich, Zurich, Switzerland. 15Department of Neurology, University
of Utah School of Medicine, Salt Lake City, Utah, USA. 16Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, USA. 17Istanbul
University, Istanbul Medical Faculty, Medical Genetics, Millet Caddesi, Capa, Fatih, Istanbul, Turkey. 18Developmental Pediatrics, University of Hawaii at Manoa,
Honolulu, Hawaii, USA. 19Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA. 20Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, Texas, USA. 21Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA. 22Department of Medicine, Baylor College
of Medicine, Houston, Texas, USA. 23Center for Human Genetics, Bioscientia, Ingelheim, Germany. 24Department of Human Genetics, Rheinisch-Westfälische
Technische Hochschule (RWTH) University of Aachen, Aachen, Germany. 25Inserm U-983, Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France. 26Molecular Medicine Unit, Institute of Child Health, University College London, London, UK. 27Department of Neurosciences, Howard Hughes Medical Institute,
University of California, San Diego, La Jolla, California, USA. 28Département de Génétique et INSERM U-781, Hôpital Necker-Enfants Malades, Université Paris
Descartes, Paris, France. 29Howard Hughes Medical Institute and Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA. 30Wilmer Eye
Institute and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Correspondence should be
addressed to N.K. ([email protected]).
Received 15 November 2010; accepted 22 December 2010; published online 23 January 2011; corrected after print 29 March 2011; doi:10.1038/ng.75 6
© 2
01
1 N
atu
re A
me
ric
a, In
c. A
ll r
igh
ts r
es
erv
ed
.
NATURE GENETICS VOLUME 43 | NUMBER 3 | MARCH 2011 189
ARTI CL ES
Genetic and functional studies have shown that defects in genes
encoding components of the ciliary apparatus lead to an overlap-
ping set of clinical phenotypes that include retinal degeneration,
renal cystic disease, polydactyly and other skeletal abnormalities,
fibrosis of various organs, and a complex range of anatomical and
functional defects of the central and peripheral nervous system. The
recognition of the clinical overlap between discrete clinical entities
attributable to ciliary dysfunction has led to the unification of such
disorders under the ciliopathy module1,2. This integration has also
been reflected in the genetic architecture of ciliopathies: although
TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum
Erica E Davis1,2, Qi Zhang3, Qin Liu3, Bi ll H Diplas1, Lisa M Davey1, Jane Hartley4, Corinne Stoetzel5,
Katarzyna Szymanska6, Gokul Ramaswami7, Clare V Logan6, Donna M Muzny8, Alice C Young9,
David A Wheeler8, Pedro Cruz9, Margaret Morgan8, Lora R Lewis8, Praveen Cherukuri9, Baishali Maskeri9,
Nancy F Hansen9, James C Mullikin9, Robert W Blakesley9, Gerard G Bouffard9, NISC Comparative
Sequencing Program9, Gabor Gyapay10, Susanne Rieger11, Burkhard Tönshoff11, I lse Kern12,
Neveen A Soliman13, Thomas J Neuhaus14, Kathryn J Swoboda15,16, Hulya Kayseri li17, Tomas E Gallagher18,
Richard A Lewis19–22, Carsten Bergmann23,24, Edgar A Otto7, Sophie Saunier25, Peter J Scambler26,
Philip L Beales26, Joseph G Gleeson27, Eamonn R Maher4, Tania Attié-Bitach28, Hélène Dollfus5,
Colin A Johnson6, Eric D Green9, Richard A Gibbs8, Friedhelm Hildebrandt7,29, Eric A Pierce3 &
Nicholas Katsanis1,2,30
Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is
underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here
we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated
nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a
large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro
evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes
pathogenic alleles to ~5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with
diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by
functional analysis of all variants informs the genetic architecture of inherited disorders.
1Center for Human Disease Modeling, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA. 2Department of Pediatrics,
Duke University Medical Center, Durham, North Carolina, USA. 3F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania, USA. 4Department of Medical and Molecular Genetics, Institute of Biomedical Research, University of Birmingham, Birmingham, UK. 5Laboratoire de Génétique Médicale EA3949, Avenir INSERM, Université de Strasbourg, Strasbourg, France. 6Section of Ophthalmology and Neurosciences, Leeds
Institute of Molecular Medicine, St. James’s University Hospital, Leeds, UK. 7Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA. 8Human
Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA. 9National Institutes of Health Intramural Sequencing Center, National Human Genome
Research Institute, National Institutes of Health, Bethesda, Maryland, USA. 10Genoscope Centre National de Séquençage, Crémieux, Evry, France. 11University
Children’s Hospital, Heidelberg, Germany. 12Department of Pediatrics, University Hospital of Geneva, Switzerland. 13Department of Pediatrics, Kasralainy School of
Medicine, Cairo University, Cairo, Egypt. 14Division of Nephrology, University Children’s Hospital Zurich, Zurich, Switzerland. 15Department of Neurology, University
of Utah School of Medicine, Salt Lake City, Utah, USA. 16Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, USA. 17Istanbul
University, Istanbul Medical Faculty, Medical Genetics, Millet Caddesi, Capa, Fatih, Istanbul, Turkey. 18Developmental Pediatrics, University of Hawaii at Manoa,
Honolulu, Hawaii, USA. 19Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA. 20Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, Texas, USA. 21Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA. 22Department of Medicine, Baylor College
of Medicine, Houston, Texas, USA. 23Center for Human Genetics, Bioscientia, Ingelheim, Germany. 24Department of Human Genetics, Rheinisch-Westfälische
Technische Hochschule (RWTH) University of Aachen, Aachen, Germany. 25Inserm U-983, Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France. 26Molecular Medicine Unit, Institute of Child Health, University College London, London, UK. 27Department of Neurosciences, Howard Hughes Medical Institute,
University of California, San Diego, La Jolla, California, USA. 28Département de Génétique et INSERM U-781, Hôpital Necker-Enfants Malades, Université Paris
Descartes, Paris, France. 29Howard Hughes Medical Institute and Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA. 30Wilmer Eye
Institute and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Correspondence should be
addressed to N.K. ([email protected]).
Received 15 November 2010; accepted 22 December 2010; published online 23 January 2011; corrected after print 29 March 2011; doi:10.1038/ng.756
© 2
011
Na
ture
Am
eri
ca
, In
c. A
ll r
igh
ts r
es
erv
ed
.
« Ciliome » in ciliopathies
Sophie Saunier Tania Attié-Bitach U781 Valérie Cormier-Daire U781 Jean-Michel Rozet U781
Renal ciliopathies (NPH and associated syndromes)
Fetal ciliopathies (Meckel, Hydrolethalus)
SRP syndromes (EVC, JATD) Retinal ciliopathies (LCA)
1209 ciliary candidate genes
J. Cohen, CNRS Gif s/YvetteB. Durand, Université LyonP. Jackson, Genentech, CA
Cohorte-Necker >800 families
~50% patients with unknown
mutation
« Ciliome » in ciliopathies
1209 ciliary candidate genes
J. Cohen, CNRS Gif s/YvetteB. Durand, Université LyonP. Jackson, Genentech, CA
Cohorte-Necker >800 familles
~50% patients with unknown
mutation
Multiplexing/Capture et Sequencing
Barcoded
Multiplexing of 32 samples /run on HiSeq2500-Illumina: 4 days
Multiplexing/Capture sequencing
(Next generation sequencing)
High-Seq2500 Illumina
128 samples in a single run
Linkage analysis(Affymetrix 250k)
- ESRD 3 years- retinal degeneration- cholestasis- situs inversus
P. Nitschké, Paris Descartes Bioinformatics Platform
Ciliome analysis using Polyweb
Ciliome results Variant nb
Total variants 6194
Exonic + Splice sites 1421
Non-synonymous + frameshifts 850
Unknown SNP (dbSNP132, 1K genome, EVS) 140
variants (in-house database) 44
Quality filter (>= 5 seq et >20% seq variant) 33
Recessive model- compound heterozygous variants 4 (2 genes)
Linkage analysis 2 (1 gene)
Selection filter of the candidat variants
TTC21B
P. Nitschké, Paris Descartes Bioinformatics Platform
Validation of the mutations by Sanger sequencing
Segregation analysis in the family
c.1231C>Tp.R411Xhet
c.626C>Tp.P209Lhet
Polyphen2/SIFT
TTC21B gene
Ciliome analysis using Polyweb
- ESRD 3 years- retinal degeneration- cholestasis- situs inversus
Linkage analysis(Affymetrix 250k)
TTC21B
271 patients
NPH/cystic kidneys
Eye
CNS
Liver
Skeletal
Situs inversus
Heart
183 patients 88 patients
Senior-Løken syndrome
Joubert syndrome
Liver fibrosis Mainzer-Saldino, Jeune and
Sensenbrenner syndromes
« Ciliome » sequencing in patients with NPH or NPH-related syndromes
Isolated NPH Syndromic NPH
NGS multiplex sequencing
SyndromicNPH
IsolatedNPH
NPH/cystic kidneys
Eye
CNS
Liver
Skeletal
Situs inversus
Heart
67%56%
Isolated NPH Syndromic NPH
0
20
40
60
80
100
120
140
160
180
No mutation
Heterozygous mutation
Recessive mutation
271 patients
« Ciliome » sequencing in patients with NPH or NPH-related syndromes
> Mutations in know ciliopathy genes: Diagnostic in 43% of cases
> Identification of new genes (20%)
NPHP1
2011 - 2017
Next GenerationSequencing approach
NPHP13/WDR19/IFT144
NPHP15/CEP164
IFT140
NPHP16/ANKS6
NPHP17/IFT172
NPHP18/CEP83
NPHP19/DCDC2
IFT54/TRAF3IP1
NPHP20/MAKBP1
NPHP2/INVS
NPHP3
NPHP4
NPHP5/IQCB1
NPHP6/CEP290
NPHP7/GLIS2
NPHP8/RPGRIP1L
NPH9/NEK8
NPHP10/SDCCAG8
NPHP11/TMEM67
NPHP12/TTC21B/IFT139
NPHP1
NPHP genes17%
Unknown mutation
58%
NPHP1
NPHP genes36%
Unknown mutation
40%
NPH cohort-Necker2016
(>900 families)
High genetic heterogeneity
Autosomal recessive
Genetic heterogeneity (> 20 NPHP genes)1997 - 2011
Positional cloningcandidat gene
approaches
Genetic diagnosis in NPH
NPHP1 deletion
25% patients
Ciliome
45% of patients
Whole Exome
15% of patients
∆NPHP125%
Unknown75%
15% of patients
without genetic
diagnosis
Whole Genome
sequencing
0
20
40
60
80
100
120
isolated syndromic
bi allelic het no
Syndromic NPHIsolatedNPH
Ciliome sequencing
Am J Hum Genet, 2017
Ciliopathy protein networks
IFT-AComplex
IFT-AComplex
An
tero
gra
de
IFT
-B
Kin
esin
II
Re
trog
rad
eIF
T-A
Dyn
ein
2
Basal
Body
Transition zone:
NPHP1/4/5/6/8;; MKS3
Axo
nem
e
IFT particles
Kinesin II
Dynein 2
Ciliary
component
Ciliary
membrane
Transition fibers:
CEP164, CEP83
Inversin compartment
NPHP2/3, NEK8, ANKS6
IFT-B
IFT172
IFT54
IFT-A
IFT144
IFT140
IFT139
IFT122
IFT121
IFT43
DYNC2H1
NPHP-JBTS-MKS complex
NPHP-JBTS-MKS complex
BBSome
• Ciliary protein targeting• Ciliary protein composition• Intraflagellar transport
Assembly/composition of ciliaSignaling (Wnt/PCP, Hedgehog…)
NPHP
SDCCAG8
CEP164
INVS
IQCB1NPHP
4XPNPEP3
CEP83
MAPKBP1
GLIS2
ANKS6
NEK8
IFT52
ANKS3 FAN1
SLC41A1
TRAF3IP1
IFT140
IFT43
WDR19
TTC21B
WDR60
IFT172
SMS
JATD
IFT80
CEP120
WDR35
IFT122
DYNC2H1
WDR34
TCTEX1D2
ZNF423
AHI1
NPHP1
B9D1RPGRIP1LTMEM
67NPHP
3
CSPP1
SLS
ALMS1
OFD
CEP290
CEP19CCDC28B
WDPCP
MKS
JBTSLCA
BBS
ARL6
BBIP1BBS1
BBS7
IFT27
BBS10
BBS5
BBS12
TTC8
BBS9
TRIM32
BBS2
MKKS
BBS4
RPGRIP1
MKS1
B9D2EXOC
4TMEM
237
TMEM231
CC2D2A
TCTN2
TCTN1
TCTN3
TMEM216
OFD1C5
ORF42
TMEM138
POC1BARL
13B
INPP5ECEP41
PDE6D KIF7
TBS1D32
DDX59C2
CD3
SCLT1LZTFL1
SRP
Skeletal ciliopathies
NPH, Joubert, Meckel syndromes Bardet-Biedlsyndrome
0
2
4
6
8
10
Nu
mb
er o
f ge
nes
15%
3%
11%
4%
3%
19%
19%
26%
NPHP
IFT-A
BBS
TZ
New candidate genes
1 htzpathogenicmutation
undetected mutation
Identification of new genes with the ciliome
BBSome
IFT-AComplex
An
tero
grad
e IF
T-B
Kin
esin
II
Retro
grade
IFT-AD
ynein
2
Basal bodymicrotubules
Axo
ne
me
IFT80
NPHP-JBTS-MKS complex
IFT particle
Kinesin II
Dynein 2
CiliarycomponentCiliarymembrane
Golgi apparatus
7 pts2pts
7 pts
13 pts 5
pts
9 pts
5 pts
1pt
Ciliopathy genes
Ciliome strategy
2 mutations
Diagnostic
2 mutations 1 mutation
2nd mutational event: exon deletion hetzcDNA expression
Additional mutation in otherpatients with similar
phenotype
Fonctional studies
Fibroblastes de patients Mutants zebrafish
New candidate geneKnown gene
45% 16% 19%
No mutation
20%
Whole exome
New ciliary genes
Or
modifying effect
candidatgenes
Chemical components screening
(Biophenics)
Cohorte de patients NPH
Patient fibroblasts
3D cultures
Zebrafish TALEN models
Physiopathologicmechanisms
Zebrafish mutants
Identification of new ciliopathies genes
Diagnostic
Thérapeutic
Ciliome v31222 genes, 5.3 Mb
exome total v5> 20 000 génes, 51
Mb
34%41%
Tubulo-glomerular feedback
Na
NKCC2-Cre mice with IFT88-∆/flox mice
Deletion of the cilia in the Macula Densa
PNAS 2014
Model of FSS- regulated modulation of
apical endocytosis in proximal tubule
• Cataract
• Hypotonia
• Fanconi syndrome
• Mental retardation
• Platelet dysfunction
• …/…
• OCRL1 (chr X)
• An Inositol 5-phosphatase
(also mutated in Dent disease)
Oculo-cerebro-renal syndrome
A « new » ciliopathy
E. FilholP. KrugC. JeanpierreA. BizetV. GrampaF. LegendreM. DelousC. HumbertA. BenmerahM. FaillerM. MaciaM.C. GublerN. Hellman
Imagine/Necker:T. AttiéJM RozeV. Cormier-DaireF. TerziN. Garcelon
Necker Hospital:Services de NéphrologieService de GénétiqueReference center MARHEA
R. Salomon
L. Heidet
Plateforme GénomiqueChristine Bole, M. ParisotPlateforme BioinformatiqueP. Nitschké, C. Masson
B. Knebelman
S. BurteyU1163-Inserm
E. Huynh CongS. WoenerO. GribouvalO. BoyerK. Tory
Tous les cliniciens en France et ailleurs
S Schneider-Maunoury (Paris)A Le Bivic (Marseille)S Christensen (Copenhagen)H Arts (Nijmegen)F Hildebrandt (Ann Harbor)
BBSome
IFT-AComplex
Short ribs - polydactylyand related disorders
NPH, Joubert, Meckel syndromes
Bardet-Biedlsyndrome
Ciliary proteins networkA
nte
rogr
ade
IFT-
BK
ines
inII
Re
trograd
eIFT-A
Dyn
ein2
Basal Body
microtubules
Transition zone:NPHP1/4/8/NPHP5/6
IFT particle
Kinesin II
Dynein 2A
xon
em
e
© AB
IFT80
NPHP-JBTS-MKS complex
• Cila assembly• Ciliary proteins transport (IFT)• Signal transduction (Shh, Wnt/PCP)
An
tero
grad
eIF
T-B
Kin
esi
nII
Re
trograd
eIFT-A
Dyn
ein
2
Basal Body
microtubules
Transition fibers
IFT particle
Kinesin IIDynein 2CiliarycomponentCiliarymembrane
Axo
ne
me
Primary cilium – sensory cilium
Tranzition zone:NPHP
Cell cycle dependant
Séquençage du « ciliome » chez les patients avec ciliopathies
1200 gènes candidats ciliaires dont 60 gènes
“diagnostiques”
NPHP
IFTA
BBS
TZ
New candidate
genes
22
17
20
7
8
22
181 htz
pathogenic mutation
no mut
NPHP3
NPHP4 *CEP290IQCB1NEK8
SDCCAG8TMEM67CEP164
AHI1
DYNC2H1IFT140
TTC21B/IFT139 *
WDR19/IFT144
WDR35/IFT121
BBS1BBS12BBS2BBS5BBS7
OFD1TCTN3
TMEM237B9D1
• Bonne « efficience » (pathologies avec très
grande hétérogénéité génétique) :
Ciliome >>> Sanger
`
• Problèmes avec les résultats négatifs :
Nombreuses variations de signification
inconnue (parfois bi-allélique)
Comment rendre un résultat négatif ?