Mechanisms of Limb Formation and Associated Phenotypes · Mechanisms of Ectrodactyly anterior I II III IV V V IV I Disruption of AER-signaling Loss of distal structures. Max Planck

Max Planck Institute for Molecular Genetics

Mechanisms of Limb Formation

and Associated Phenotypes

Stefan Mundlos

Institut für Medizinische Genetik und Humangenetik

Charité, Universitätsmedizin Berlin, Berlin

Max Planck Institut für Molekulare Genetik, Berlin


GeneticDisease

Clinical Genetics

Diagnosis

Genetics

Gene Function

Molecular Pathology

Charité

MPIMG

FG Developmentand Disease

Institute forMedical Genetics

Genome

Phenotype


syndactyly

finger-like thumb

brachydactyly

Malformations of the Limbs

polydactyly

radial ray defect ectrodactyly

oligodactyly


Patterning the Limb

proximodistal

AER

FGFs

anterior-

posterior

Shh

dorso-

ventral

Wnt7a

Lmx1

en1


The Zone of Polarising Activity (ZPA)

- control of anterior-posterior pattern -


Sonic Hedgehog (Shh) mediates the ZPA signal

E10.5

E11.5

Shh co-localizeswith the ZPA


The degree of digit duplication is dosage dependent

Pathogenesis of

Polydactyly?


Mutations within the

Hedgehog pathway

Polydactyly phenotype

Cephalopolysyndactyly

(Greig syndrome)

Mutations in GLI3

Polydactyly


Mechanisms of Polydactyly

- Zone of polarising activity (ZPA)

Expression of Shh

anteriorI

II

III

IVV

"doubledose"

polydactyly

- ectopic expression of Shh -


The Apical Ectodermal Ridge (AER)

- control of proximo-distal pattern/outgrowth -


Removal of AER Results in Truncation

FGFs are

the AER

signal

FGF4


Dlx5/6-/- Mouse as a Model for Split-Hand-Foot Malformations

• Distal-less Homeoboxgenes Dlx5/6

Robledo et al, Genes Dev, 2002

E 18.5

Dlx5/6 -/-wt

E 14.5 Dlx5/6 -/-wt


Dlx5/6-/- Mouse Shows Loss of AER-Markers

Robledo et al, Genes Dev, 2002

• Loss of medial AER

Structures

• Dlx5/6 important for

maintenance of AER

Fgf8

Msx2

Dlx5/6 -/-wt


Split-Hand-Foot Phenotypes

- mutations in p63 -

P63 controls devlopment of ectodermal structures


Mechanisms of Ectrodactyly

anteriorI

II

III

IVV

V

IV

I

Disruption of

AER-signaling

Loss of

distal

structures


Dorso-ventral Polarity

- specified by signal from the ectoderm (Wnt7a)

Wnt7a

Lmx1


Lmx1 is Essential for Dorsal Pattern

Mutations in LMX1

causeNail-Patella syndrome

Lack of dorsal structures in Lmx1

knock out mice


Breakdown of Signaling Centers Results in Truncations

Mutation in WNT7

Loss of:• distal structures

• asymmetry

• nails

• dorso-ventral pattern


Limb Identity

Arm vs. Leg


Pedigree with Abnormal Wrists and ElbowsPedigree with Abnormal Wrists and Elbows

Liebenberg Syndrome


Control

ap

Patient

laterallaterallateral

patient control


Arm to Leg TransformationArm to Leg Transformation

associated with rearrangements at the PITX1 locusassociated with rearrangements at the PITX1 locus

Pitx1 is expressed in hind limb only

Family 1

Family 2

400 kbPitx1

Deletion


Family 1

Family 2

400 kbPitx1

Regulatory Elements at the DeletionRegulatory Elements at the Deletion


Family 1

Family 2

Pitx1

Enhancer Adoption at the PITX1 locusEnhancer Adoption at the PITX1 locus

Deletion

MalteSpielmann

Barrier


Transgenic expression of Pitx1 under Hs1473 enhancer Transgenic expression of Pitx1 under Hs1473 enhancer

results in forelimb to hindlimb transformationresults in forelimb to hindlimb transformation


Molecular Determinants of Hindlimb/Forelimb IdentityMolecular Determinants of Hindlimb/Forelimb Identity

Minguillon and Logan 2005

Pitx1 Tbx4 Hindlimb


Genetic Testing

in congenital disease

Genetic disease or Thalidomide?

hypoplastic thumbs absent thumbs


1000

10000

100000

1000000

10000000

100000000

1 3 6 9 12 16 18 21 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97100

103

106

109

112

115

118

121

124

Sequencing Cost / Genome

Roche 454Solexa/Illumina

SOLiDLifeTech.

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011´01 ´12

€1K

€10K

€100K

€1M

€10M

€100M

www.genome.gov/sequencingcosts

Illumina HiSeq X ten


Applications of NGSin genetic diagnostics

Gene Panel Diagnostics

all genes that are associated with a disease / phenotype

Lipid metabolismImmunologye Cardiology Deafness

Osteoporosis Skelettal dysplasiaCDG

90 Genes

27 Genes 96 Genes 90 Genes

76 Genes 167 Genes

188 Genes

currently established

HPO

2776 Genes


Skeletal Dysplasia Panel – Design

ACANACP5

ACVR1ADAMTSL2AGAAGPSALX1

ALX4ANO5ANTXR2ARSBARSE

BMP2BMPERBMPR1BCANT1

CC2D2ACDH3CEP290CHST14CHST3

COG1COL10A1COL11A1COL11A2COL2A1

COL9A1COL9A2COL9A3COMPCREBBP

CTSACUL7

DDR2EFNB1

EIF2AK3EP300ESCO2EVCEVC2

EXT1EXT2FAM58AFBXW4FGD1

FGF10FGF9FGFR1FGFR2

FGFR3FLNAFLNBFMN1FUCA1

GALNSGDF5GDF6GLB1GLI3

GNASGNPATGNPTABGNPTGGNS

GPC6GREM1

GUSBHDAC4

HOXA11HOXA13HOXD13HSPG2ICK

IDSIDUAIFT122IFT43IFT80

IHHIL1RNINPPL1KAT6B

KIF22LBRLFNGLIFRLMBR1

LMX1BLPIN2LRP4MATN3MESP2

MGPMKS1MMP13MMP9DLL3

DYM

DYNC2H1EBP

MNX1MSX2MYCNNAGLUNEK1

NEU1NFIXNIPBLNKX2-3NLRP3

NOGNPR2NSD1NSDHL

OBSL1OFD1PAPSS2PCNTPDE4D

PEX7PITX1POLR1CPOLR1DPOR

PRKAR1APTH1RPTHLHPTPN11RAB23

RAB33B

DYNC2H1EBP

MNX1MSX2MYCNNAGLUNEK1

NEU1NFIXNIPBLNKX2-3NLRP3

NOGNPR2NSD1NSDHL

OBSL1OFD1PAPSS2PCNTPDE4D

PEX7PITX1POLR1CPOLR1DPOR

PRKAR1APTH1RPTHLHPTPN11RAB23

RAB33B

RECQL4RMRP

ROR2RPGRIP1LRUNX2SALL1SALL4

SBDSSGSHSH3BP2SH3PXD2BSHH

SHOXSLC17A5SLC26A2SLC35D1

SMARCAL1SOX9SUMF1TBCETBX15

TBX3TBX4TBX5TCOF1THPO

TMEM67TP63TRAPPC2TRIP11TRPS1

TRPV4

TWIST1WDR35

WISP3WNT3WNT7A

167 genesTarget region of 658 kbpCriteria: Causative for monogenic skeletal dysplasias + dysostoses no predominant connective tissue involvement or abnormal bone massno lysosomal storage disorder (group 27 of nosology)


Excellent coverage:Mapped reads: 2153679Unmapped reads: 84020Fraction with cov. >= 10 : 0.9854506Fraction with cov. >= 20 : 0.9839304

Skeletal Dysplasias and Dysostoses Panel

Paired-end 150 bp sequencing on MiSeq


Skeletal Dysplasia Panel – Case 13-0812

Clinic: lethal chondrodysplasia


Analysis of Results


FLNB:NM_001164317:exon2:c.517G>A:p.A173T

Fetus with Skeletal DysplasiaAnalysis of Data

Mutation in filamin B:

Boomerang Dysplasia

Atelosteogenesis type I

child

father

mother


Peter RobinsonTomasz Zemojtel

Malte Spielmann

Claus-Eric Ott

Peter Krawitz

Jochen Hecht

Sebastian Köhler

Uwe Kornak

Mateusz Kolanczyk

Acknowledgements

Funded by

Berlin National & international cooperators:

Niels Benatar (Braunschweig, Germany)

Regina Betz (Bonn, Germany)

Peter Meinecke (Hamburg, Germany)

Ingo Kurth (Jena, Germany)

Thomy de Ravel (Leuven, Belgium)

Anna Rajab (Muscat, Oman)

Jeske van Harssel (Utrecht, Netherlands)

Andrew O.M. Wilkie (Oxford, UK)

Francesco Brancati (Rome, Italy)

Paola Ferrari (Modena, Italy)

Ulrich Mennen (Pretoria, South Africa)

Niki Foulds (Wessex, UK)


Vielen Dank für Ihre

Aufmerksamkeit!

Documents

Mechanisms of Limb Formation and Associated Phenotypes · Mechanisms of Ectrodactyly anterior I II III IV V V IV I Disruption of AER-signaling Loss of distal structures. Max Planck