2008 Barcelona - European Society of Human Genetics

Concurrent Sessions
transgenic mice overexpressing FRG1, a gene proximal to the deletion,
showed a phenotype resembling the FSHD disease . However, increased
expression of FRG1 in FSHD patients has not been a uniform
finding, and up to now several studies have failed in identifying the
molecular mechanism affecting the FSHD locus functionality .
We took advantage of ChIP/MeDIP and 3D immuno-FISH assays as
complementary approaches to depict the higher order of chromatin organization
of 4q35 .2 region during myogenic differentiation of healthy
and FSHD myoblast and mesoangioblast stem cells . We found that
FRG1 undergoes to muscle specific regulation through a two-step
activation mechanism, whereby removal of H3-K27 methylation and
Polycomb complex components precedes MyoD recruitment on the
FRG1 promoter; intriguingly, the same chromatin structure and PcG
recruitment were contemporaneously found on D4Z4 array, rendering
the Polycomb complex the first molecular player that links FSHD locus
to myogenic differentiation . Moreover, D4Z4 H3-mK27 signals were
strongly reduced in FSHD myoblasts in respect to controls, suggesting
the severe impairment of the PcG complex recruitment .
Nevertheless, molecular alterations of the D4Z4 array do not have in
FSHD myoblasts an effect in cis on FRG1 gene expression . These
observations evidence a role of 4q35 D4Z4 in muscle differentiation,
probably through inter-chromosomal interactions .
c12.3
Active transport of the ubiquitin ligase miD1 along the
microtubules is regulated by protein phosphatase 2A
B. Aranda Orgilles 1 , J. Aigner 1 , R. Schneider 1,2 , S. Schweiger 1,3 ;
1 Max Planck Institute for Molecular Genetics, Berlin, Germany, 2 Institute of
Biochemistry, Innsbruck, Austria, 3 Division of Pathology and Neuroscience,
Ninewells Hospital, Dundee, United Kingdom.
Mutations in the MID1 protein have been found in patients with Opitz
BBB/G syndrome (OS), which is characterised by multiple malformations
of the ventral midline . MID1 is a microtubule-associated protein
that stabilizes microtubules and, in association with the regulatory subunit
of protein phosphatase 2A (PP2A), α4, provides ubiquitin ligase
activity for the ubiquitin-specific modification of PP2A. Using Fluorescence
Recovery After Photobleaching (FRAP) technology, we show
here that MID1 is actively and bi-directionally transported along the microtubules,
and that this movement is directly linked to its MAP kinase
and PP2A-mediated phosphorylation status . Intact transport depends
on both kinesins and dyneins and is inhibited upon taxol and colcemide
treatments . MID1 proteins carrying missense mutations in the
α4 binding domain still bind the microtubules but can not be actively
transported. Likewise, knock-down of the α4 protein, inhibition of PP2A
activity by okadaic acid and fostriecin or the simulation of permanent
phosphorylation at Ser96 in MID1 stop the migration of MID1-GFP,
while preserving its microtubule-association . In summary, our data uncover
an unexpected and novel function for PP2A, its regulatory subunit
α4 and PP2A / α4 / mTOR signalling in the active transport of the
MID1 ubiquitin ligase complex along the cytoskeleton . Furthermore,
a failure in the microtubule directed transport of this protein complex
would be an attractive mechanism underlying the pathogenesis of OS
in patients with B-box1 mutations .
c12.4
A centrosomal protein molecularly links Usher syndrome to
Leber congenital amaurosis and Bardet-Biedl syndrome in the
retina
H. Kremer 1,2 , E. van Wijk 1,3 , F. Kersten 3,1 , N. Zaghloul 4 , T. Peters 1 , A. Kartono 3,2 ,
S. Letteboer 3,2 , U. Wolfrum 5 , N. Katsanis 4 , R. Roepman 3,2 ;
1 Department of Otorhinolaryngology, Radboud University Nijmegen Medical
Centre, Nijmegen, Netherlands, 2 Nijmegen Centre for Molecular Life Sciences,
Radboud University Nijmegen, Nijmegen, Netherlands, 3 Department of Human
Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands,
4 Departments of Ophthalmology and Molecular Biology and Genetics,
McKusick Nathans Institute of Genetic Medicine, Johns Hopkins University
School of Medicine, Baltimore, MD, United States, 5 Department of Cell and
Matrix Biology, Institute of Zoology, Johannes Gutenberg University of Mainz,
Mainz, Germany.
Usher syndrome (USH) is the most common cause of hereditary deafblindness
in man . Ten loci are known for Usher syndrome, and we and
others provided evidence for the existence of a protein network of the
USH proteins at different subcellular sites in the retina and inner ear .
Disruption of one of the members of the USH network can lead to malfunction
and degeneration of both photoreceptor cells and hair cells .
To elucidate the pathogenic mechanisms of Usher syndrome, we
searched for novel interacting partners for the intracellular region of
USH2A isoform B . This revealed the interaction with a centrosomal
protein . Interestingly, simultaneous screens for interactors of the recently
identified lebercilin (LCA5, and associated with Leber congenital
amaurosis, LCA), identified the same centrosomal protein. In order to
clarify the role of this protein in vivo, knockdown studies in zebrafish
were performed . This gave rise to a classical planar cell polarity phenotype,
similar to the defects observed after knockdown of the genes
involved in Bardet-Biedl syndrome (BBS) . Yeast two-hybrid interaction
analysis subsequently revealed a specific physical interaction between
the centrosomal protein and BBS6 .
Our data indicate that the same centrosomal protein interacts physically
with USH2A, lebercilin and BBS6, thereby linking the retinal ciliopathies
Usher syndrome, Leber congenital amaurosis and Bardet-Biedl
syndrome at the molecular level . The physical and genetic interactions
between proteins/genes involved in Usher syndrome and Bardet-Biedl
syndrome suggest a putative role for the Usher interactome in the establishment
of planar cell polarity, with a central role of the cilia .
c12.5
study of the role of the Ofd1 transcript in limb patterning and
endochondral bone development
S. Bimonte 1 , L. Quagliata 1 , R. Tammaro 1 , M. Ascenzi 2 , B. Franco 1,3 ;
1 Telethon Institute of Genetics and Medicine-TIGEM, Naples, Italy, 2 Department
of Orthopaedic Surgery, Biomechanics Research Division, University of California,
Los Angeles, CA, United States, 3 Department of Pediatrics, University
Federico II, Naples, Italy.
Oral-facial-digital type I (OFDI) syndrome is an X-linked dominant male
lethal developmental disorder characterized by oral, facial and digital
abnormalities . Recent data ascribed OFDI to the growing number of
diseases due to dysfunction of primary cilia . Ofd1 null mutants recapitulate
the phenotype observed in OFDI patients and displayed skeletal
defects . To overcome the embryonic male and perinathal female lethality
observed in Ofd1 null mutants we have generated a conditional
model with Ofd1 limb mesenchyme specific inactivation. These mice
displayed a severe polydactyly with loss of antero-posterior digit patterning,
aberrant cilia formation, and shortened long bones . Defective
digit pattering was found to be associated to progressive loss of Shh
signaling and impairment of Gli3 processing . Shortening of long bones
was found to be associated to misregulation of Ihh expression and activity
during endochondral bone formation as revealed by RNA in situ
studies . Immunoistochemical analyses to assess the proliferative state
of chondrocytes revealed an increase in the number of proliferating
pre- and hypertrofic chondrocytes in male mutants. This data suggest
that the shortening of long bones observed in Ofd1fl|Prx1Cre mice is
likely due to an increase in the number of proliferating chondrocytes
associated to a delay in terminal chondrocytes differentiation . Finally
Von kossa staining and RNA in situ studies demonstrated defective
bone mineralization accompanied by loss/reduction of bone collar development
suggesting a defect in osteoblast differentiation .
Altogether our data demonstrate that Ofd1 is a pattering factor that
plays multiple roles in limb and endochondral bone development .
c12.6
integration into molecular diagnostic procedures of systematic
screening for sequence variants of unknown significance using
a splicing reporter minigene
M. Vezain 1 , I. Tournier 1 , A. Martins 1 , C. Bonnet 1 , S. Krieger 2 , S. Baert-Desurmont
1 , A. Killian 1 , A. Hardouin 2 , T. Frébourg 1,3 , M. Tosi 1 ;
1 Inserm U614, Faculty of Medicine, Rouen, France, 2 Laboratory of Clinical and
Oncological Biology, Centre François Baclesse, Caen, France, 3 Department
of Genetics, University Hospital, Institute for Biomedical Research, Rouen,
France.
Unclassified variants (UVs) found in genes involved in genetic diseases
may have an effect on pre-mRNA splicing . In clinical practice,
the interpretation of UVs is limited because patient blood samples
are often not suitable for RNA analysis . We have recently developed
a screening strategy based on genomic DNA from patients, using a
splicing reporter minigene . We have now applied this screening protocol
to more than 150 UVs from many genes including MSH2, MLH1,