2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
and to detect new sequence variants within SPG4 gene in 51 HSP
patients in Estonia . Fifty healthy individuals were used as controls .
The majority of patients had pHSP and only five patients had cHSP
form of the disease . cHSP was the most frequent in sporadic cases
. All 51 samples were screened with DHPLC and abnormal elution
profiles were sequenced. Nine changes (G609A, A810G, 1299-1g>c,
1310delA, 1370+215g>c, 1370+202delG, C1503A, 1477-1481del-
GAGAA, 1966insA) in SPG4 gene were detected showing no gender
predisposition . Seven were new and only found in 11 HSP patients,
but two (one new - 1370+215g>c and other previously described -
1370+202delG) were detected both in patients and controls . Only two
mutations (1299-1g>c, C1503A) showed familial segregation, in which
three and two family members were affected, respectively . Other mutations
were found in individuals from different families . In conclusion
we associate above-mentioned seven new mutations with 11 AD-HSP
cases, since new sequence variants were found only in patients compared
to healthy controls . The rest of the patients must be checked for
other changes (larger INDELs) in SPG4 and other genes involved in
this disease should be considered for further analysis .
P05.187
in depth investigation of -1 frameshifting in expanded cAG
repeat tracts using time-lapse live cell imaging
S. J. Stochmanski 1 , C. Gaspar 1 , D. Rochefort 1 , J. Laganiere 1 , P. Hince 1 , G. Di
Cristo 2 , G. A. Rouleau 1 ;
1 Centre of Excellence in Neuromics, CHUM Research Centre, Montréal, QC,
Canada, 2 Centre de Recherche, Hôpital Ste-Justine, Montréal, QC, Canada.
Spinocerebellar ataxia type 3 (SCA3) results from an expansion of a
polyglutamine-encoding CAG tract in the ATXN3 gene . We have previously
demonstrated that this expanded CAG tract is subject to -1 ribosomal
frameshifting into the alanine frame, which seems to confer an
increased toxicity, and that the antibiotic anisomycin reduces both -1
frameshifting and cell toxicity . Currently, dual-tagged ATXN3 reporter
constructs were created to express DsRED in the main (glutamine)
frame and EGFP in the -1 (alanine) reading frame, and these reporter
constructs contained either 14 CAG repeats, 89 CAG repeats, or 92
CAA repeats . Constructs were transfected into COS-1 cells as well as
mouse cortical organotypic slice culture preparations, and were monitored
for the production of red or green fluorescence signals using
time-lapse live-cell two-wave fluorescent microscopy. Employing this
technique, we have confirmed the occurrence of -1 frameshifting for
the CAG89 construct, whereas the constructs bearing wild-type CAG
or expanded CAA repeats did not show significant frameshifting. We
also determined that there is a marked time delay between the onset of
glutamine-containing protein expression and the production of frameshifted
species, as well as a correlation between frameshifitng and cell
death. These findings argue in favour of local glutamine codon starvation,
followed by a shift in the reading frame to resume translation of
the protein in the alanine frame and seem to confirm the implication of
-1 ribosomal frameshifting in the pathogenesis of SCA3 .
P05.188
Quantitative analysis of SMN gene based on real-time PcR
M. Šimášková 1 , H. Poláková 2 , L. Kádaši 1,2 ;
1 Comenius University, Faculty of Natural Sciences, Department of Molecular
Biology, Bratislava, Slovakia, 2 Institute of Molecular Physiology and genetics
Slovak Academy of Science, Bratislava, Slovakia.
Spinal muscular atrophy (SMA) is an autosomal recessive disorder,
caused by the homozygous absence of the survival motor neutron
gene (SMN1) in approximately 94% of patients . Since most carriers
have only one SMN1 gene copy, several quantitative analyses have
been used for the SMA carrier detection . We performed a SMN1quantitative
real-time PCR analysis using an allele specific primer for the
carrier detection of SMA. We compared the sensitivity, specificity,
advantages and disadvantages recently described in the quantitative
method, using TaqMan probes and a newly developed Plexor TM technology,
which had not previously been used for identifying heterozygotes
. Using a comparative threshold cycle (C T ) method and the DNA
fragment of human beta globine gene as a reference gene, the gene
copy number of SMN1 was quantified. The sensitivity and specificity
of the TaqMan and Plexor TM technologies were similar; moreover,
the assay efficiency was almost ideal when using the Plexor TM technology
. The incidence of SMA (1:6000-10000) and the notable carrier
frequency (1:35-50) as well as the severity of disease, and the lack of
effective treatment may justify the implementation of such analysis in
DNA diagnostic laboratories .
P05.189
molecular Genetics and Epidemiology in spanish
spinocerebellar Ataxia
H. San Nicolás 1 , J. Corral 1 , I. Banchs 1 , L. De Jorge 1 , O. Combarros 2 , J. Berciano
2 , C. Serrano 3 , M. Calopa 4 , A. Matilla 5 , D. Genís 6 , V. Volpini 1 ;
1 Center for Molecular Genetic Diagnosis of Hereditary Diseases (CDGM)- IDI-
BELL, Hospitalet de Llobregat, Spain, 2 Dep of Neurology. Hosp Univ Marqués
de Valdecilla, Santander, Spain, 3 Dep of Neurology. Hosp Sant Joan de Deu,
Martorell, Spain, 4 Dep of Neurology. Hosp Univ De Bellvitge. IDIBELL, Hospitalet
de Llobregat, Spain, 5 Health Sciences Research Institute Germans Trias
i Pujol, Badalona, Spain, 6 Dep of Neurology. Hosp Univ Josep Trueta, Girona,
Spain.
Autosomal dominant cerebellar ataxias (ADCA) are a clinically and
genetically heterogeneous group of neurodegenerative disorders in
which several spinocerebellar ataxia (SCA) genes have been cloned:
SCAs1-3, SCAs6-7, SCA12 and SCA17; sharing a CAG repeat expansion
mutations which generally encodes a polyglutamine tract . In
SCA8 the mutation is an untranslated CTG repeat . We have analyzed
340 unrelated familial and 1,013 sporadic and idiopathic cases of SCA .
Over the familial cases 6 .04% were SCA1; 26 .85% SCA2; 32 .89%
SCA3; 7 .38% SCA6; 6 .71% SCA7; 14 .77% SCA8; and 1 .34% SCA17 .
In 22 familial index cases with SCA8 expansions the allele range goes
from 85 to 470 repeats (129 .36% ± 67 .55%; Pearson Coef . =52 .22%) .
Maternal transmissions presented elongations of the CTG combined
sequence ranging from +2 to +13 repeats (7 .50 ± 5 .5; Pearson Coef .
= 73 .33%) . In contrast, paternal transmissions presented contractions
ranging from 1 to -17 repeats (-6 .83 ± 7 .51; Pearson Coef = -
109 .93%) . Several giant SCA8 expansions ranges from 401 to 1,126
(N= 9), carried by unaffected adult individuals and being originated
from homozygous SCA8 females with alleles of moderate size . In contrast,
the homozygous males have transmitted contracted alleles, as in
heterozygous cases occurs . We have tested 90 individuals from general
population and the distribution of SCA8 alleles could be classified
in two groups: 15 to 34 CTGs, with frequency 98% and 77 to 86 CTGs,
with frequency 2% . About 60% of familial ADCA cases remained genetically
unclassified. No SCA mutations were detected in the 1,013
isolated and idiopathic cases of spinocerebellar ataxia .
P05.190
Novel mutations found in ABcA4 in spanish families
J. Aguirre-Lamban1,2 , R. Riveiro-Alvarez1,2 , D. Cantalapiedra1,2 , M. Garcia-
Hoyos1,2 , E. Vallespin1,2 , A. Avila-Fernandez1,2 , J. Gallego-Merlo1,2 , M. Lopez-
Martinez1,2 , M. Trujillo-Tiebas1,2 , C. Ramos1,2 , C. Ayuso1,2 ;
1 2 Fundacion Jimenez Diaz, Madrid, Spain, Centro de Investigacion Biomedica
en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain.
Introduction: ABCA4 mutations have been associated with Stargardt
disease (STGD) . A few cases with autosomic recessive cone-rod dystrophy
(arCRD) and autosomic recessive retinitis pigmentosa (arRP)
have also been found to have ABCA4 mutations . Comparative genetic
analyses of ABCA4 variation and diagnostics have been complicated
by substantial allelic heterogeneity .
Subjects and Methods . 31 unrelated families, were previously studied
with the ABCR400 genotyping microarray . In patients with either none
or only one mutant allele were analysed by dHPLC, sequencing and
multiplex ligation-dependent probe amplification (MLPA). Haplotype
analysis was also performed .
Results . 27 ABCA4 mutations were found in 31 Spanish patients with
the ABCR400 microarray. We confirmed that the p.Arg1129Leu mutation
is the most frequent in Spanish patients. dHPLC allowed us to find
eleven novel mutations and were not found in the 100 chromosomes .
Using both tools, the mutation detection rate obtained was incremented
in a 24 .2% with respect to the use of the microarray alone . We
detected 1 .6 % of false positives and 1 .6 % of false negatives . In 17/31
patients (54 .8%) in which the second or neither mutation was found by
these methodologies, they were studied with MLPA; however no deletion
or duplication was found in these samples . No mutation was found
in 4/31 patients (12 .9%) .
Conclusions . The ABCR400 microarray is a comprehensive screening
tool for genetic variation in patients with ABCR-associated retinal pa-