2008 Barcelona - European Society of Human Genetics

Concurrent Sessions
c07.1
Oligosaccharyltransferase subunits mutations in non-syndromic
mental retardation
F. Molinari 1 , F. Foulquier 2 , P. S. Tarpey 3 , W. Morelle 2 , J. Teague 3 , S. Edkins 3 , P.
A. Futreal 3 , M. R. Stratton 3 , G. Turner 4 , G. Matthijs 5 , J. Gecz 6 , A. Munnich 1 , L.
Colleaux 1 ;
1 INSERM U781 and Université Paris Descartes, Paris, France, 2 UMR CNRS/
USTL 8570, Lille, France, 3 Wellcome Trust Sanger Institute, Cambridge, United
Kingdom, 4 The Gold Service, Adelaide, Australia, 5 Laboratory for Molecular
Diagnostics, Center for Human Genetics, Leuven, Belgium, 6 Department of Pediatrics
and School of Molecular & Biomedical Science, University of Adelaide,
Adelaide, Australia.
Mental Retardation (MR) is the most frequent handicap among children
and young adults . While a large proportion of X-linked MR genes
have been identified, only four genes of autosomal recessive non-syndromic
MR (AR-NSMR) have been described so far .
Here, we report on two new genes involved in autosomal and X-linked
NSMR. First, autozygosity mapping in two sibs born to first cousin
French family led to the identification of a region on 8p23.1-p22. This
interval encompasses the gene N33/TUSC3 encoding one subunit of
the oligosaccharyltransferase (OTase) complex which catalyses the
transfer of an oligosaccharide chain on nascent proteins, the key step
of N-Glycosylation . Sequencing N33/TUSC3 identified a one basepair
insertion, c .787_788insC, resulting in a premature stop codon,
p .N263fsX300, and leading to mRNA decay . Surprisingly, glycosylation
analyses of patient fibroblasts showed normal N-glycan synthesis and
transfer . Subsequently, screening the X-linked N33/TUSC3 paralog,
the IAP gene, identified a missense mutation (c.932T>G, p.V311G) in
two brothers with X-linked NSMR . Interestingly, quantitative RT-PCR
analyses showed increased IAP expression in N33/TUSC3 mutated
cells, suggesting that normal N-glycosylation observed in patient fibroblasts
may be due to functional compensation .
Recent studies of fucosylation and polysialic acid modification of
neuronal cell adhesion glycoproteins have shown the critical role of
glycosylation in synaptic plasticity. However, our data provide the first
demonstration that a defect in N-Glycosylation can result in NSMR . Altogether,
our results demonstrate that fine regulation of OTase activity
is essential for normal cognitive function development, providing therefore
new insights to understand the pathophysiological bases of MR .
c07.2
Influence of Friedreich ataxia GAA non-coding repeats
expansions on pre-mRNA processing
F. Pagani;
ICGEB, Trieste, Italy.
The intronic GAA repeat expansion in the frataxin gene causes the
hereditary neurodegenerative disorder Friedreich’s ataxia . While it is
generally believed that GAA repeats block transcription elongation, a
direct proof in eukaryotic system is lacking . We tested in hybrid minigenes
the effect of GAA and TTC repeats on nascent transcription
and pre mRNA processing . Unexpectedly, disease-causing GAA repeats
(n=100) did not affect transcriptional elongation in nuclear HeLa
RUN ON assay nor pre mRNA transcript abundance but resulted in a
complex defect on pre mRNA processing . GAA but not TTC repeats
insertion downstream of reporter exons resulted in their partial or complete
exclusion from the mature mRNAs and in the generation of a
variety of aberrant splicing products . Interestingly, the GAA expansion
induced the accumulation of an upstream pre mRNA splicing intermediate,
which is not turned over into mature mRNA . This effect of GAA
repeats was observed to be position and context-dependent, as their
insertion at different distance from the reporter exons had a variable effect
on splice site selection . Reduction of GAA triplets partially restored
normal splicing consistent with a repeat length dependent phenotypic
variability .
This data indicates, for the first time, an association between GAA
non-coding repeats and aberrant pre-mRNA processing and suggests
an alteration of the coordination between transcription and pre-mRNA
processing in this disease . Transcribed GAA repeats might create a
“decoy” exon that binding to trans acting splicing factors may interfere
with normal turnover of non-coding intronic RNA leading to degradation
and lower transcript levels .
c07.3
mechanisms of mEcP2 function underlying Rett syndrome as
revealed from overexpression and knock-down systems in vitro
M. Vecsler 1,2 , A. J. Simon 1,3 , G. Rechavi 1,3 , N. Amariglio 1,3 , E. Gak 1,2 ;
1 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 2 Genetics Institute,
Sheba Medical Center, Tel Hashomer, Israel, 3 Cancer Research Center,
Institute of Hematology, Sheba Medical Center, Tel Hashomer, Israel.
Rett syndrome is a severe X-linked neurodevelopmental disorder
mainly affecting girls and is the second common cause of mental retardation
in girls after the Down syndrome . The methyl CpG-binding
protein 2 (MeCP2), a ubiquitous transcriptional repressor interacting
with the chromatin remodeling machinery, is considered a single
causative factor of Rett syndrome and related phenotypes, and some
autistic cases . Our present study is focusing on interaction between
MeCP2 and chromatin proteins leading to changes in chromatin architecture
and silencing of gene expression . Using confocal microscopy,
we demonstrate that MeCP2 protein is localized at the nuclear heterochromatin
compartment together with the heterochromatin protein 1
alpha (HP1α). In addition, we have developed in-vitro systems overexpressing
the normal MeCP2 and MeCP2 containing Rett related mutations,
as well as a knock-down system of the endogenous MECP2
using specific siRNA. Studying these systems with a comprehensive
gene regulation antibody array, we demonstrate that the expression of
a specific set of nuclear proteins, including hBRM/hSNF2a component
of SWI/SNF, HMGB1 high mobility group protein, G9a histone methyltransferase,
PRMT1 protein arginine methyltransferase and HDAC2
histone deacetylase, is synchronized with MeCP2 overexpression and
knock-down . Moreover using co-immunoprecipitation analyses, we
demonstrate a direct interaction between MeCP2 and hBRM/hSNF2a
component of ATPase-dependent SWI/SNF complex involved in global
chromatin remodeling mechanism. Our findings suggest that MeCP2
acts through parallel mechanism of chromatin remodeling involving
HDACs and SWI/SNF complex, thereby inducing local as well as large
scale changes in chromatin architecture and compaction .
c07.4
mutations in UBE are associated with X-linked infantile
spinal muscular atrophy (XL-smA) and cause decreased gene
expression in patients and carrier females
A. Meindl1 , J. Ramser1 , C. Lenski1 , M. von Rhein2 , B. Wirth3 , K. O. Yariz4 , M. E.
Ahearn4 , L. Baumbach-Reardon4 ;
1Department of Obstetrics and Gynecology, Technical University Munich,
Munich, Germany, 2University Children’s Hospital Mainz, Mainz, Germany,
3 4 Institute of Human Genetics, University of Cologne, Cologne, Germany, Miller
School of Medicine, University of Miami, Miami, FL, United States.
X-linked infantile spinal-muscular atrophy (XL-SMA; MIM301830) is a
rare X-linked disorder that presents with the clinical features of hypotonia,
areflexia and multiple congenital contractures (arthrogryposis)
associated with anterior horn cell loss and infantile death . Large scale
mutation analysis in the linkage interval (DXS8080-DXS7132) resulted
in the detection of three rare novel variants in exon 15 of the gene
coding for the Ubiquitin-Activating Enzyme E1 (UBE1): two missense
mutations (c .1617 G>T, p .Met539Ile, c .1639 A>G, p .Ser547Gly) present
each in one XL-SMA family and one synonymous C>T substitution
(c.1731 C>T, p.Asn577Asn) identified in additional four unrelated
families . Each of these variants was demonstrated to segregate with
the disease . Absence of the missense mutations was demonstrated
for 3550, absence of the silent mutation was shown in 7914 control
X-chromosomes. These results yielded statistical significant evidence
for the association of the silent substitution and the two missense mutations
with XL-SMA (P = 2 .416x10-10 , P = 0 .001815) . We have also
demonstrated that the silent C>T substitution leads to significant reduction
of UBE1-expression in patients and interestingly to a lesser extent
also in carrier females and alters the methylation pattern of exon
15, implying a plausible role of this DNA element in developmental
UBE1- expression in humans . UBE1 catalyzes in the ubiquitin-proteasome
system (UPS) the first step in ubiquitin conjugation to mark cellular
proteins for degradation . Our observations indicate that XL-SMA
is part of a growing list of neurodegenerative disorders associated with
defects in the ubiquitin-proteasome pathway .