2008 Barcelona - European Society of Human Genetics

Clinical genetics
P01.215
the ctG repeat expansion size correlates with the splicing
defects observed in muscles from myotonic dystrophy type 1
patients
A. Botta1 , F. Rinaldi1 , C. Catalli2 , L. Vergani3 , E. Bonifazi2 , V. Romeo3 , E. Loro3 ,
A. Viola1 , C. Angelini3 , G. Novelli1,2 ;
1 2 Tor Vergata University of Rome, Rome, Italy, Tor Vergata Hospital, Rome,
Italy, 3University of Padoa, Padoa, Italy.
Myotonic dystrophy type 1 (DM1; MIM#160900) is caused by an unstable
(CTG)n repetition located in the 3’UTR of the DMPK gene . Untranslated
expanded DMPK transcripts are retained in ribonuclear foci
and sequester CUG-binding proteins essential for the maturation of
pre-mRNAs . In this study, we investigated the effects of CTG expansion
length on three molecular parameters associated with the DM1
muscle pathology: 1) the expression level of the DMPK gene; 2) the
degree of splicing misregulation and 3) the number of ribonuclear foci .
To this purpose, we selected 6 muscle biopsies from DM1 patients
with an expansion below 500 repetitions, 6 muscle samples from DM1
patients carrying a mutation above 1000 CTGs and 6 controls muscle
samples . Splicing analysis of the IR, MBNL1, c-TNT and CLCN1 genes
demonstrated that the level of aberrant splicing isoforms is strikingly
different between the two groups of DM1 muscle samples . In addition,
a significant correlation was observed in the extent of abnormal splicing
and the CTG repeat length for all the genes studied . RNA-FISH
analysis reveals that the number of ribonuclear foci accumulating in
DM1 muscle sections increases in patients with a higher (CTG)n number.
On the contrary, we did not find any relationships between the
expression level of the DMPK gene transcript and average expansion
sizes . These data indicate that the CTG repeat length plays a key role
in the extent of splicing misregulation and foci formation, thus providing
a useful link between the genotype and the molecular cellular phenotype
in DM1 .
P01.216
the results of a systematic screening for new mutations in NAiP
gene
P. Apostol, D. Cimponeriu, M. Stavarachi, N. Butoianu, I. Minciu, C. Burloiu, M.
Toma, D. Cheta, C. Serafinceanu, L. Gavrila;
Institute of Genetics, Bucharest, Romania.
Background: The NAIP gene is located in an inverted duplicated region
(5q13), enclosed to some repetitive elements which predispose
to chromosomal rearrangements . However, a small number of NAIP
mutations/polymorphisms were reported in this region which is considered
a hot-spot mutational . The most common NAIP mutation is a
deletion involving exon 5, which has been reported to be associated
with spinal muscular atrophy (SMA) complications .
Aim: The aim of this study was to screen the NAIP gene for new mutations/polymorphisms
.
Material and methods: We selected for this study SMA patients (n=63),
dialyzed patients (n=200) and healthy clinical subjects (control group,
n=370) after informed consent obtaining . The DNA samples for all subjects
were screened for presence of NAIP exon 5 by PCR . We also
used specific molecular methods to test the presence of additional mutations
in this region .
Results and discussion: The homozygous absence of NAIP exon 5
was observed in 20,6% SMA patients and in 1% of dialyzed and control
subjects . The restriction pattern (DraI) and the indirect methods
have been shown that a sample from dialyzed lot has an abnormal
electrophoretic and melting comportment . The sequencing analyses
have confirmed a heterozygous state for a G/T substitution (Arginine/
Serine), which creates a new restriction site for DraI endonuclease .
The etiology of renal failure could not be established .
Conclusion: In this study we have identified a new coding G/T SNP in
NAIP gene and the highest frequency of homozygous NAIP exon 5
deletion in SMA patients (20,6%) .
P01.217
soluble expanded PABPN1 exacerbates cell death in
Oculopharyngeal muscular Dystrophy
C. Messaed 1,2 , P. Dion 1 , A. Abu-Baker 1 , D. Rochefort 1 , J. Laganiere 1 , B. Brais 1 ,
G. A. Rouleau 1 ;
1 Center of excellence in neuromics, Notre-Dame-CHUM, Montreal, Canada,
QC, Canada, 2 University of McGill, Montreal, QC, Canada.
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant
late-onset myopathy characterized by progressive eyelid drooping,
swallowing difficulty and proximal limb weakness. OPMD results
from the expansion of a polyalanine repeat (GCG)8-13 in exon 1 of
the poly(A)binding protein nuclear1 (PABPN1) gene . Nuclear aggregates
consisting of tubular filaments within muscle fibers of OPMD
patients are the pathological hallmark of the disease . These aggregates
contain expanded PABPN1 (expPABPN1), poly(A)RNA as well
as components of the ubiquitin-proteasome degradation pathway and
molecular chaperones . Whether nuclear aggregates are pathogenic or
simply the consequence of a molecular defense mechanism remains
controversial in OPMD and in the field of neurodegenerative disorders.
To evaluate the contribution of nuclear aggregates to cellular toxicity,
we first targeted molecular mechanisms known to interfere with exp-
PABPN1 aggregation . Our cellular model shows that increasing the
availability of nuclear soluble expPABPN1 significantly exacerbates
cell death . We also used time lapse imaging to follow the evolution of
cells overexpressing expPABPN1 without interfering with any cellular
pathway. Cells with nuclear aggregates show a significantly prolonged
lifespan compared to cells harbouring a diffusely distributed soluble
expPABPN1. This is the first report indicating the beneficial effect of
nuclear aggregation in OPMD . The formation of nuclear aggregates
may reflect an active process by which cells sequester and inactivate
the soluble toxic form of expPABPN1 . The structural change in exp-
PABPN1, induced by the pathogenic alanine expansion, may lead to a
gain of aberrant protein interactions, or alternatively prevent normally
occurring interactions to take place .
P01.218
spinal muscular atrophy in University Hospital Brno, czech
Republic- Genetic counselling, DNA analysis, Prenatal analysis
R. Gaillyová1 , E. Zapletalová1 , L. Fajkusová1 , Š. Prášilová1 , J. Šoukalová1 , I.
Valášková1 , Z. Kalina2 , A. Michenková2 , E. Makaturová2 ;
1University Hospital Brno, Masaryk University Brno, Brno, Czech Republic,
2University Hospital Brno, Brno, Czech Republic.
Spinal muscular atrophy (SMA) is the second most frequent lethal autosomal
recessive disease in Europeans with the incidence of 1/6000
-10 000 and a carrier frequency of 1/40-50 . SMA is in approximately
96% of cases caused by homozygous deletion of the SMN1 gene . 4%
of SMA patiens have a combination of the deletion or conversion in
one allele and an intragenic mutation on the second one . The SMAdetermining
gene (survival motor neuron - SMN), is present on 5q13
in two copies, a telomeric SMN1 gene and a cetromeric SMN2 gene,
which are highly homologous and contain only five base-pair differences
. However, increased SMN2 gene copy number, which can occur
as the result of gene conversion events, is associated with a milder
SMA phenotype .
Since 1999, we have performed the DNA analysis of SMA, the carrier
detection from 2003 and from 2005 we analysed the copy number of
SMN2 gene .
Until now, we examined more then 300 SMA patients, only one SMN1
copy was detected in 17 of them, we identified 6 point mutations.
Currently, we perform the genetic counselling in families with SMA occurrence,
we offer DNA analysis of the SMN1 gene in the proband and
the detection of SMA carriers . We offer the DNA analysis of SMN1
gene for partners of SMA carriers . In families with high risk of SMA
in the child we offer prenatal DNA diagnostics from cultivated amnial
cells .
Supp . by 1A/8608-4, Molecular aspects of diagnostics and therapy of
spinal muscular atrophy, IGA MH CR
P01.219
Accuracy of marker analysis, quantitative real time PcR and
Multiple Ligation-dependent Probe Amplification to determine
smN2 copy number in patients with spinal muscular atrophy
L. Alias, M. J. Barcelo, S. Bernal, E. Also-Rallo, R. Martínez, M. Baena, P. Gallano,
M. Baiget, E. F. Tizzano;
Hospital Sant Pau, Barcelona, Spain.
Spinal muscular atrophy (SMA) is an autosomal recessive disease
characterised by degeneration of motor neurons of the spinal cord and
is caused by mutations in the SMN1 gene . The SMN2 gene is the
highly homologous SMN1 copy that is present in all patients . There is