2009 Vienna - European Society of Human Genetics

Molecular basis of Mendelian disorders
gene, encoding spastin. SPG4-HSP and generally described as a
pure form of the disease_that is, as spastic paraparesis often associated
with a decreased sense of vibration in the lower limbs and urinary
problems. However, it is characterized by a large variability in the age
at onset (ranging from early infancy up to the eighth decade). Patients
usually have a family history of the disease but some may also present
as sporadic cases due to incomplete penetrance or censor effects. In
the latter, non-genetic causes of spastic paraparesis, such as primary
progressive multiple sclerosis, (PPMS) are usually searched for.. We
report 3 patients with a primary diagnosis of PPMS, based on MRI
findings, who carried a SPG4 mutation. Interestingly, 2 out of the 3
patients had a family history of gait disorder or clinical diagnosis of
MS and in one, the father died at age 60 (censor effect). The remaining
patient fulfilled Mc Donald’s criteria (MRI and CSF) for MS. Those
findings must lead to important caution with PPMS diagnosis not to
understimate genetic spastic paraplegia because of consequences for
appropriate genetic counselling. Those results could also modify our
understanding of the mechanisms of demyelinating diseases and of
the interactions between axonal degeneration and inflammation.
P12.155
molecular analysis of spinal muscular atrophy in iranian
population
S. Vallian, N. Noori;
The University of Isfahan, Isfahan, Islamic Republic of Iran.
Spinal muscular atrophy (SMA) is an autosomal recessive genetic
disorder of motor neurons. Defects in genes for survival motor neuron
(SMN) and neural apoptosis inhibitory protein (NAIP) have been
shown to be associated with the disease. Among the genetic defects,
deletions in exon 7 and 8 of SMN as well as exons 4 and 5
of NAIP gene were found to be most significant. In this study, deletions
in SMN and NAIP genes were examined in 35 unrelated SMA
patients (14 type I, 5 type II, 16 type III) patients. Deletion frequency
in SMN (E7 and 8) and NAIP (E4 and 5) in patients with SMA type I
was 92.8%,42.8%,64.3%,78.6%; in type II, 60%,20%,80%,80%; and
in type III, 16.6%,6.25%,43.7%,43.7%, respectively. About 7.1% of patients
with SMA type I; 20% with type II and 56.2% with type III showed
no deletions for the exons examined. Moreover, homozygous deletion
in E7 and/or 8 of the SMN gene was found in 62.5% of the patients,
with high frequency in both type I (78.5%) and type II (80%), and less
frequency (48.5%) in type III. Similarly, homozygous deletions of E4
and/or E5 of NAIP gene was highest in type I (92.9%) compare to
type II (60%) and type III (6%). Our data suggest a strong association
of deletions in E4 and E5 of NAIP together with deletions in SMN (E7
and 8) with more severe form of SMA (SMA type I and II) in Iranian
population.
P12.156
Estimation of smN2 copy number in 536 unrelated spanish smA
patients by Multiplex-Ligation dependent Probe Amplification
(mLPA)
S. Bernal 1,2 , L. Alías 1,2 , M. J. Barceló 1,2 , E. Also-Rallo 1,2 , R. Martínez-Hernández
1,2 , F. J. Rodríguez-Alvarez 3,4 , E. Aller 5,6 , E. Grau 5,6 , A. Peciña 7,8 , L. Ortiz 7,8 ,
M. J. Rodríguez 1,2 , P. Gallano 1,2 , S. Borrego 7,8 , J. M. Millán 5,6 , C. Hernández-
Chico 3,4 , E. F. Tizzano 1,2 ;
1 Servicio de genética. Hospital de la Santa Creu i Sant Pau, Barcelona, Spain,
2 CIBERER, Barcelona, Spain, 3 Unidad de genética. Hospital Ramón y Cajal,
Madrid, Spain, 4 CIBERER, Madrid, Spain, 5 Unidad de genética. Hospital La
Fe, Valencia, Spain, 6 CIBERER, Valencia, Spain, 7 Unidad de gestión clínica
de genética, reproducción y medicina fetal. Hospital Virgen del Rocío, Sevilla,
Spain, 8 CIBERER, Sevilla, Spain.
Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by
absence or mutations in the SMN1 gene. SMA patients are classified
into 3 groups, type I (the most severe), type II (the intermediate form)
and type III (the less affected) according to age of onset, achieved motor
abilities, and life span. SMN2 is the SMN1 highly homologous copy
that is considered as a modifier of the disease.
We estimated the SMN2 copy number in 536 unrelated SMA patients
from different Spanish centres. We employed the Multiplex-Ligation
dependent Probe Amplification (MLPA) technique, which includes a
mixture of specific probes for the SMA locus.
The majority of the type I patients showed two copies of the SMN2
gene (209/261,80%), type II patients presented mainly 3 SMN2 copies
(126/150, 84%), whereas 91% (114/125) of type III patients have 3
(67%) or 4 (24%) SMN2 copies. These results confirm that SMN2 copies
are strongly related to disease severity. However, the correlation is
not absolute (i.e. 36 type I patients showed three SMN2 copies and 9
type III patients had two copies). This study performed in a large cohort
of subjects, allowed us to improve the genetic characterisation of the
SMA locus. Moreover, it will be useful to define a subtype of patients
(i.e. those with three SMN2 copies and different SMA type) to further
investigate the functional copies of SMN2 and possible modifiers of
the phenotype. Supported by GENAME Project, CIBERER and FIS
05-2416.
P12.157
High throughput, complete genotyping of the tNFRsF1A gene.
this project is supported through coordination theme 1 (Health)
of the European community‘s FP7
S. Lezer, A. Yakir, N. Navot;
Pronto Diagnostics Ltd, Rehovot, Israel.
The TNF Receptor Associated Periodic Syndrome (TRAPS) is a
rare autosomal dominant multisystematic autoinflammatory disorder,
caused by sporadic mutations in the TNF super family Receptor 1A
gene (TNFRSF1A). Genetic diagnosis of TRAPS consists usually of
sequencing of exons 2-4 of TNFRSF1A gene. However, mutations
which cause TRAPS have been also found outside these exons; therefore,
a system that would offer complete genotyping of the TNFRSF1A
gene would be highly advantageous. We are currently developing an
answer to this need using a new, high throughput technique, for simultaneous
detection of both known and novel point mutations (SNPs and
pathogenic mutations) and of large-scale genetic rearrangements in
the TNFRSF1A gene. This system, called EMMA (enhanced mismatch
mutation analysis) is based on electrophoretic heteroduplex analysis
(HDA) and semiquantitative multiplexed PCR by multi-capillary electrophoresis.
The EMMA (Fluigent, France) mutation detection method
is an alternative to dHPLC and sequencing. It combines all the advantages
of the screening before sequencing strategy: high throughput,
major reduction in sequencing costs and high productivity. The
presence of both point mutations and long-range rearrangements has
a “signature” that can be associated with quantitative and objective
numerical criteria. The analysis of electrophoregrams can be fully automated;
a feature that for the rare TRAPS case is less imperative, but
will be very useful for high-throughput screening of clinically important
genes with more prevalent SNPs and pathogenic allele variants.
P12.158
New trinucleotide diseases analysis based on HPLc and QF PcR
M. Skrzypczak-Zielinska 1,2 , A. Sulek-Piatkowska 3 , A. Plawski 2 , R. Slomski 2 , U.
G. Froster 1 ;
1 Institute of Human Genetics, Leipzig, Germany, 2 Institute of Human Genetics,
Polish Academy of Sciences, Poznan, Poland, 3 Department of Genetics, Institute
of Psychiatry and Neurology, Warszawa, Poland.
Trinucleotide expansions are an important mutational form specifically
in neurogenerative disorders such as HD (Huntington disease), different
types of SCA (Spinocerebellar ataxia), SBMA (Spinal and bulbar
muscular atrophy) and DM1 (Myotonic dystrophy type 1). Trinucleotide
disorders are characterized by an increasing of severity of disease
course in the next generation of mutation curriers. The number
of repeats may reach over 2000 in severe affected individuals. These
larger expansions require specific and time consuming methods for
identification, such as fragments lengths analysis by polyacrylamide
gel electrophoresis (PAGE) or Southern blotting. Identification and exact
determination of alleles on the molecular level is very important for
diseases diagnosis and prognosis. The aim of our study was to develop
a highly sensitive, automated and economical molecular method
for determination and characterization of trinucleotide repeat regions.
Here we present the molecular test based on high performance liquid
chromatography (HPLC) and quantitative fluorescent (QF) PCR
for characterization of trinucleotide repeats. We analyzed 403 samples
- clinically diagnosed with different trinucleotide disorders. Our
results indicated high accuracy and consistency of the data obtained
with HPLC (± 3 CAG) compared to classical methods based on PAGE
and sequencing. The results obtained using combined QF and Long
PCR showed higher precision in comparison to the results obtained
by traditional techniques. We conclude that HPLC, QF and Long PCR