2009 Vienna - European Society of Human Genetics

Plenary Lectures
PL2.2
massive parallel sequencing of ataxia genes after array-based
enrichment
A. Hoischen 1 , C. Gilissen 1 , W. van der Vliet 1 , P. Arts 1 , N. Wieskamp 1 , S. Vermeer
1 , R. Meijer 1 , M. Buckley 1 , B. Kremer 2 , N. van Slobbe-Knoers 1 , J. Veltman 1 ,
H. Scheffer 1 ;
1 Department of Human Genetics, Radboud University Nijmegen Medical Centre,
Nijmegen, The Netherlands, 2 Department of Neurology, Radboud University
Nijmegen Medical Centre, Nijmegen, The Netherlands.
Targeting multiple disease genes by massively parallel sequencing has
tremendous diagnostic potential but requires new ‘front-end’ methods
to enrich templates to be sequenced. Here we validated the arraybased
sequence capture method for medical sequencing approaches
in heterogeneous genetic disorders. As a model disease we chose
autosomal recessive ataxia and selected 4 patients with known mutations
in ataxia genes. Genomic sequences of all known disease genes,
including all intronic sequences and 5kb up- and downstream of each
gene, as well as a novel ataxia candidate locus were represented on a
single oligonucleotide array, comprising 2.5 Mb of genomic sequence
in total. After enrichment each of the patients DNA was analyzed by
one quarter of a Roche GS FLX Titanium sequencing run, resulting
in approximately 100 Mb of genomic sequence per patient. This was
sufficient to reach an average per base coverage of 34-fold in all targeted
regions. Enrichment showed high specificity, as up to 88% of all
reads mapped uniquely to the targeted regions. Very few single reads
mapped to non-targeted regions, interestingly mainly to chr.9_random,
which might indicate that this region represents a minor allele in the
existing human genome assembly. Importantly, this approach enabled
an unbiased detection of all known mutations in our 4 patients, deletions
and point mutations, as well as known SNP variants (hetero- and
homozygous). These results show that massive parallel sequencing of
enriched samples enables tailor-made genetic diagnosis of heterogeneous
disorders.
PL2.3
the sLc29A3 gene is mutated in Pigmented Hypertrichosis with
insulin Dependent Diabetes mellitus syndrome and interacts
with the insulin signalling pathway
S. T. Cliffe1 , J. M. Kramer2 , K. Hussain3 , J. H. Robben2 , E. K. de Jong2 , A. P. de
Brouwer2 , E. Nibbeling2 , E. Kamsteeg2 , M. Wong4 , J. Prendiville5 , C. James3 ,
R. Padidela3 , C. Becknell6 , H. van Bokhoven2 , P. M. T. Deen2 , R. C. M. Hennekam3
, R. Lindeman1 , A. Schenck2 , T. Roscioli2,7 , M. F. Buckley2,1 ;
1 2 South Eastern Area Laboratory Services, Sydney, Australia, Nijmegen Centre
for Molecular Life Sciences, Nijmegen, The Netherlands, 3Institute for Child
Health, London, United Kingdom, 4Children’s Hospital, Westmead, Sydney,
Australia, 5British Columbia’s Children’s Hospital, Vancouver, BC, Canada,
6 7 Dermatology Associates of Kentucky, Lexington, KY, United States, Sydney
South West Integrated Genetics Service, Sydney, Australia.
Pigmented hypertrichotic dermatosis with insulin dependent diabetes
(PHID) syndrome is a recently described autosomal recessive disorder
associated with predominantly antibody negative, insulin-dependent
diabetes mellitus. In order to identify the genetic basis of PHID
and study its relationship with glucose metabolism we performed homozygosity
mapping in five unrelated families followed by candidate
gene sequencing. Five loss of function mutations were identified in the
SLC29A3 gene which encodes a member of a highly conserved protein
family that transports nucleosides, nucleobases and nucleoside
analogue drugs, hENT3. We show that PHID is allelic with a related
syndrome without diabetes mellitus, H syndrome. The interaction of
SLC29A3 gene with insulin signaling pathways was then studied using
an established model in Drosophila melanogaster. Ubiquitous knockdown
of the Drosophila ortholog of hENT3, dENT1 is lethal under stringent
conditions whereas milder knockdown induced scutellar bristle
phenotypes similar to those previously reported in knockdown of the
Drosophila ortholog of the Islet gene. A cellular growth assay showed
a reduction of cell size/number which could be rescued or enhanced
by manipulation of the Drosophila insulin receptor and its downstream
signaling effectors, dPI3K and dAkt. In summary, inactivating mutations
in SLC29A3 cause a syndromic form of insulin dependent diabetes
in humans and in Drosophila profoundly affect cell size/number
through interactions with the insulin signaling pathway. These data
suggest that the further investigation of the role of SLC29A3 in glucose
metabolism is a priority for diabetes research.
PL2.4
Duplication of conserved non-coding sequence elements
- a novel mechanism in the pathogenesis of congenital
malformations
E. Klopocki 1 , K. Dathe 1 , A. Brehm 2 , K. W. Kjaer 3 , C. Ott 1 , I. Kurth 4 , S. Mundlos
1,5 ;
1 Institute of Medical Genetics, Charite Universitätsmedizin Berlin, Berlin, Germany,
2 Max-Planck-Institut für Molekulare Genetik, Berlin, Germany, 3 Wilhelm
Johannsen Centre for Functional Genome Research, Institute of Cellular and
Molecular Medicine, University of Copenhagen, Copenhagen, Denmark, 4 Institute
of Human Genetics, Universitätsklinikum Hamburg Eppendorf, Hamburg,
Germany, 5 Max-Planck Institut für Molekulare Genetik, Berlin, Germany.
Two thirds of the sequence conserved among mammals is not protein
coding. The precise function of such non-coding sequence elements
(CNEs) is unknown but they have been proposed to regulate time and
tissue specific gene expression over distances as great as 1Mb. Using
high-resolution array CGH we detected CNE-containing duplications
in patients with congenital malformations. We identified duplications of
1) a regulatory sequence 1Mb upstream of SHH (ZRS) in triphalangeal
thumb-polysyndactyly syndrome and in 2) Laurin-Sandrow syndrome,
3) a 5kb regulatory element 110kb downstream of BMP2 in brachydactyly
type A2, 4) 2Mb containing several CNEs upstream of SOX9 in
Cooks syndrome, and 5) 1.7Mb 5’ of MSX2 in a condition resembling
cleidocranial dysplasia. In all cases the duplications are arranged in
tandem. We show that the CNE contained in the duplication at the
BMP2 locus is an enhancer regulating BMP2 expression exclusively
in the limbs, thus, functioning as cis-regulatory element. We postulate
that duplications of cis-regulatory elements cause selective deregulation
of the target gene resulting in disturbance of dosage-dependent
signalling pathways only in those areas and/or time points that correspond
to the CNE’s regulatory potential. Our data provide the molecular
cause for so far genetically unresolved conditions (Laurin-Sandrow,
Cooks syndrome) and show genetic heterogeneity for others.
Furthermore, duplications of CNEs can be considered a novel genetic
mechanism for developmental defects. Given the importance of temporal-spatial
gene regulation during embryonic development it is to be
expected that a large number of developmental defects are caused by
mutations affecting such distant enhancers/repressors.
PL2.5
mitosis updated - PicH and the anaphase threads
T. Schwarzbraun1 , L. Wang2 , P. Ulz1 , E. A. Nigg2 , M. R. Speicher1 ;
1 2 Institute of Human Genetics, Graz, Austria, Max-Planck Institute of Biochemistry,
Martinsried, Germany.
The process of mitosis has been illustrated in more or less unchanged
ways in textbooks for decades and still resembles the original observations
made by Flemming back in 1882. However, PICH (Plk1-interacting
checkpoint helicase) was recently identified as an essential component
of the spindle assembly checkpoint and shown to localize to
kinetochores, inner centromeres, and most interestingly, it decorates
thin threads connecting separating sister-chromatids even until late
anaphase. PICH-positive threads evolve from inner centromeres as
they stretch in response to tension reaching up to 15 µm in length.
With the discovery of DNA threads connecting sister-chromatids until
late anaphase a new level of cell cycle regulation seems likely. The
properties of the PICH protein lead to the hypothesis that it associates
with catenated centromeric DNA, where it may act as a tension
sensor to monitor the bipolar attachment of sister kinetochores and
thus ensuring accurate chromosome segregation. Indeed, we could
recently demonstrate that these threads are in fact mainly constituted
of stretched alphoid centromeric DNA and that topoisomerase activity
is required during anaphase for the resolution of PICH-positive
threads. Additionally, knock-down as well as over-expression of the
PICH protein result in severe chromosomal mis-segregation. These
data indicate that the complete separation of sister chromatids occurs
later than previously assumed as well as that PICH and the alphoid
centromeric DNA repeats are part of an additional mechanism to safeguard
the genomic integrity.