European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Concurrent Symposia
were significantly less frequent in CSD melanomas. We showed that
germline variants of MC1R are a major susceptibility factor for BRAF
mutations among non-CSD melanomas. Further analysis our array
CGH data suggested a genomic region harboring KIT to be important
in melanomas on mucosa, acral skin and skin with CSD. Oncogenic
mutations or focused copy number increases in the gene were found in
40% of acral and mucosal melanomas, and 30% of CSD melanomas.
These results suggest that the existing drugs such as imatanib and
new are inhibitors against KIT could be useful for certain melanoma
types. More recent studies correlating phenotypic alterations with the
underlying genotype have shown that mutations in BRAF are associated
with distinct histopathological features of the primary tumor that
can be combined to simple algorithms that can predict the mutation
status of BRAF with high accuracy. Our data show that melanoma is
comprised of genetically and phenotypically distinct subtypes with different
requirements for treatment stratification.
S13. Comprehensive analysis of the SRY-box 10 locus (SOX10):
Implications for diseases of neural crest derivatives
B. J. Pavan;
Genetic Disease Research Branch, National Human Genome Research Institute,
National Institutes of Health, Bethesda, MD, United States.
The transcription factor SOX10 is a key regulator of genes involved
in neural-crest derived melanocytes, enteric neurons, and myelinating
Schwann cells. Mutations in the SOX10 gene are associated with human
diseases that affect these cell populations, including impaired pigmentation,
enteric aganglionosis (e.g., Waardenburg-Shah syndrome),
and demyelinating peripheral neuropathy. While SOX10 targets have
been identified, the mechanisms involved in the transcriptional regulation
of SOX10 remain elusive. Recently, we used comparative sequence
analysis to identify highly-conserved, non-coding segments
upstream of SOX10. We demonstrated that three of these segments
are encompassed by a 15-kb deletion residing ~ 50 kb upstream of
Sox10 in a mouse model of Waardenburg-Shah syndrome. Importantly,
this deletion does not completely compromise Sox10 expression
in all tissues. Thus, other sequences at the Sox10 locus are likely involved
in transcriptional regulation, and mutations in these sequences
may contribute to neural crest disorders in humans.
One goal of our lab is to fully characterize SOX10 regulatory sequences
and to establish if mutations in these sequences contribute to human
disease. Our studies involve four distinct research areas: (1) Testing
conserved segments for enhancer activity in cell lines; (2) Determining
if these conserved segments drive reporter gene expression in a
tissue-specific manner in zebrafish and mouse; (3) Deleting these conserved
segments in BACs and testing for altered reporter gene expression
in transgenic mice; and (4) Screening these conserved segments
for sequence variations in a panel of >600 DNA samples isolated from
patients with enteric aganglionosis (Hirschsprung disease). These
studies will provide important clues about the transcriptional regulation
of SOX10 and the full role of the transcription factor in human development
and disease.
S14. Cilia in Skeletal Development: identification of Evc as a
mediator of Ihh signalling
V. L. Ruiz-Perez 1 , H. J. Blair 2 , M. Rodriguez-Andres 1 , M. Blanco 3 , Y. Liu 2 , H.
Peters 2 , C. Miles 2 , J. A. Goodship 2 ;
1 Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones
Científicas, Madrid, Spain, 2 Institute of Human Genetics, International Centre
for Life, Newcastle upon Tyne, United Kingdom, 3 Department of Human Anatomy
and Embryology, Faculty of Medicine, Universidad Complutense de Madrid,
Madrid, Spain.
Cilia are projections from the cell made of a core of microtubules and
associated proteins. Cilia are classified according to their microtubule
components: 9+2 motile cilia and 9+0 primary cilia, which are usually
immotile. The importance of primary cilia in development, e.g. leftright
development, and postnatally, e.g. in the kidney, is increasingly
recognised. Intraflagellar transport (IFT) is required to generate cilia.
IFT mutants are embryonic lethal but generation of conditional alleles
enables studies regarding requirement for cilia in later processes and
have shown that they play a critical role in endochondral bone formation.
A limitation of this approach is that removal of cilia disrupts all of
their many functions. We identified two causative genes for the human
chondrodysplasia Ellis-van Creveld syndrome (EvC, OMIM:225500)
by positional cloning. We have inactivated Evc in the mouse and show
that Evc -/- mice develop an EvC-like syndrome, including short ribs,
short limbs, and dental abnormalities. LacZ driven by the Evc promoter
revealed that Evc is expressed in the developing bones and the orofacial
region. We developed antibodies that localized the Evc protein
to the base of the primary cilium. Recent studies of IFT mutants have
revealed that primary cilia are integral to Hedgehog signalling. Analysis
of the growth plate of Evc -/- mice shows delayed bone collar formation
and advanced maturation of chondrocytes. By in situ hybridization
we detected that Indian hedgehog (Ihh) is expressed normally in the
growth plates of Evc -/- mutants but expression of the Ihh downstream
genes Ptch1 and Gli1 were markedly decreased. Cilia formation is not
affected in Evc mutants and western blot analysis indicates that Gli3
processing is normal. We conclude that Evc is required for Ihh signalling
in endochondral bone formation.
S15. Molecular basis of primary ciliary dyskinesia
S. Amselem;
INSERM U.654, Hôpital Armand-Trousseau, Paris, France.
Defects in structure and function of cilia have recently been associated
with a growing number of rare diseases. The phenotypic features
of these so-called ciliopathies reflect the diverse biological properties
of these evolutionarily conserved structures that protrude from the
apical surface of most eukaryotic cells. Cilia, which contain a microtubule
cytoskeleton (axoneme) structurally related to the flagella of
spermatozoa, can be classified according to their axonemal components:
primary cilia, with sensory function, are involved in a wide range
of disorders, whereas motile cilia are involved in the most prominent
ciliopathy called primary ciliary dyskinesia (PCD). PCD is a rare respiratory
disease, affecting approximately 1/16,000 individuals, due to
impaired mucociliary clearance. This disorder, usually transmitted as
an autosomal recessive trait, is characterized by chronic airway inflammation,
male infertility, and, in approximately half of the patients,
abnormal left-right asymmetry, defining the Kartagener syndrome. The
disease phenotype results from various axonemal defects involving
the motile respiratory and embryonic nodal cilia, as well as the flagella
of spermatozoa. Its molecular basis is just beginning to be elucidated,
with, to date, 5 genes known to be involved in that condition
(i.e. DNAI1, DNAH5, DNAH11, RPGR, and TXNDC3). These results
were obtained by various means, including original candidate-gene
approaches based on data from a one-billion-year-old unicellular flagellar
algae (Chalmydomonas reinhardtii) and from see urchin. The current
challenges are to identify new PCD genes and to characterize
the molecular and cellular mechanisms involved in ciliary dyskinesia.
Flagellated protists like trypanosomes represents promising models to
achieve these goals. These lower eukaryotes possess genes for flagellar
motility whose inactivation reproduces the motility and ultra-structural
defects seen in patients with PCD. In addition, in spite of the evolutionary
distance between these unicellular organisms and humans,
the complementation of genetically modified strains of trypanosomes
with human orthologous sequences seems conceivable.
S16. Making sense of cilia: Bardet Biedl syndrome and other
ciliopathies
P. L. Beales;
The Institute of Child Health,, University College London, London, United Kingdom.
Despite knowledge of primary (non-motile) cilia for over a century, recently
ascribed functions have rekindled interest in these sessile cellular
appendages. Less than a decade ago, cilia were associated with
renal cyst formation (through observations in mouse models of polycystic
kidney disease) although the mechanism is still hotly debated.
More recently, developmental biologists have discovered the origins
of left-right asymmetry common to vertebrates and demonstrated the
involvement of specialised “spinning” primary cilia. Our own area of
study, the Bardet-Biedl syndrome along with nephronophthisis, both
causes of renal cyst formation have revealed the importance of primary
cilia in human disease.
In this lecture, I will describe the evidence for ciliary dysfunction in Bardet-Biedl
syndrome and how this is leading us to define extended roles
for cilia in developmental pathways. I will also describe an approach
we have taken to discover the causes of other diseases of cilia function
thus widening the scope of this emerging class of ciliopathies.