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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


Axo

ne

me




An

tero

grad

eIF

T-B

Kin

esi

nII

Re

trograd

eIFT-A

Dyn

ein

2

Basal Body

microtubules

Transition fibers

IFT particle


Axo

ne

me




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



node

Hamada, 2002



CILIA

3

9+0 9+2



node

Hamada, 2002



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 !

http://insects.eugenes.org/species/about/species-gallery/Drosophila_melanogaster/Drosophila_melanogaster.jpg

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


Axo

ne

me




An

tero

grad

eIF

T-B

Kin

esi

nII

Re

trograd

eIFT-A

Dyn

ein

2

Basal Body

microtubules

Transition fibers

IFT particle


Axo

ne

me




An

tero

grad

eIF

T-B

Kin

esi

nII

Re

trograd

eIFT-A

Dyn

ein

2

Basal Body

microtubules

Transition fibers

IFT particle


Axo

ne

me




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 defects √ √ √ √ √ √ √ √ √ √





√ √ √ √



√ √


√ √ √ √


i




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


√ √ √ √ √ √ √ √ √ √ √


Liver defects √ √ √ √ √ √ √ √ √ √





√ √ √ √



√ √


√ √ √ √


i

i




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


√ √ √ √ √ √ √ √ √ √ √


Liver defects √ √ √ √ √ √ √ √ √ √





√ √ √ √



√ √


√ √ √ √


i

i

i

i




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



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



















Motile cilia



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

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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


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


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


• 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


Axo

ne

me




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 ?

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