2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
P06.273
intronic sequence changes may have unpredictable effects on
splicing
F. Joncourt, S. Gallati;
Human Genetics, Department of Pediatrics, University Hospital, Berne, Switzerland.
Mutation scanning often identifies yet undescribed sequence changes
in genomic DNA, which are not easily classified as either pathogenic
or neutral solely by sequence inspection . Increasing knowledge about
the splicing process has revealed its great complexity: In addition to
the well known splice-site consensus sequences in recent years a multitude
of regulatory elements have been identified both within introns
as well as within exons. In order to assess / confirm their pathogenic
nature, we have analysed the effect on splicing of several yet functionally
uncharacterized intronic sequence changes in different genes
by analysing their respective RNA’s . Lymphocytes from patients were
immortalized, RNA was extracted and reverse transcribed . The cDNA
was then amplified by PCR and analysed by PAGE or agarose gel electrophoresis
. Bands with altered electrophoretic mobility were isolated,
purified and sequenced. Two mutations located within the conserved
donor and acceptor splice sites respectively (NF1: c .3496+1G>A and
SPG4: c .1688-2A>G) were shown to lead to exon skipping as expected.
c.357+2T>A identified in a patient’s DMD-gene, however, resulted
in the inclusion of 31 bases of intronic sequence into the mRNA leading
to a frameshift, while exon skipping would have left the reading
frame intact . This latter case further illustrates that the effect of an
unknown mutation on the splicing process can not easily be predicted .
The results emphasize the need for functional characterization of newly
described sequence changes .
P06.274
sPRY1 molecular analysis in subjects with ureteral duplicity
L. Artifoni 1 , E. Benetti 2 , S. Negrisolo 1 , S. Centi 1 , G. Caridi 3 , G. Ghiggeri 3 , L.
Murer 1,2 ;
1 Laboratory of Nephrology, Department of Paediatrics, University of Padua,
Padua, Italy, 2 Paediatric Nephrology, Dialysis and Transplantation Unit, Department
of Paediatrics, University of Padua, Padua, Italy, 3 Laboratory of Physiopathology
of Uremia, Gaslini Institute, Genoa, Genoa, Italy.
Studies on murine models demonstrated that Sprouty1 protein, encoded
by Spry1 gene, modulates Gdnf/Ret signal, which activates a crucial
gene network in urinary tract development . Spry1-knockout mice
develop supernumerary ureteric buds, that result in multiple ureters
and kidneys . In the literature, there only one report about mutational
analyses of human SPRY1 gene, the homologue of murine Spry1,
even if the gene is known to be expressed in fetal renal tissue .
We carried out mutational analysis of SPRY1 gene in patients with ureteral
duplicity: 23 isolated and 4 familial cases . On each DNA sample,
the coding region and 5’UTR were analysed by SSCP and all PCR
products were then directly sequenced (ABI PRISM 3100 Applied Biosystem)
. DNA from 6 subjects without kidney and urinary tract anomalies
was used as control group .
We detected 5 polymorphisms (SNPs), previously reported in databases,
and 1 nucleotide substitution, which has never been reported .
The frequency of this substitution was estimated in 127 umbilical cord
blood DNA samples and was 0 .094 . In order to understand SPRY1
role in renal development, mutational analysis will be extended to a
population of subjects with different malformative nephrouropathies
and association studies with the detected polymorphisms will be performed
.
P06.275
transcriptomic analysis of statin treated rat skeletal muscle
cells
M. J. Ko, H. S. Choi, H. S. Jeong, J. I. Ahn, S. Y. Kim, H. J. Chung;
National Institute of Toxicological Research of Korea, Seoul, Republic of Korea.
Statins are competitive hydroxy-3-metyl glutaryl coenzyme A(HMG-
CoA) reductase inhibitors that inhibit the synthesis of cholesterol from
mevalonic acid . Statins are the drugs most frequently used to reduce
plasma cholesterol levels and decrease cardiovascular events . However,
the side effects associated with the use of statins have been
highlighted by the withdrawal of cerivastatin from the market in 2001,
but little is currently known about the effect of statins on the RNA expression
profile of skeletal muscle cells and mechanism of myopathy.
To address this issue, we used rat L6 myoblast cells, which can differentiate
into myocytes, in this study . We treated cells with cerivaststin
which had developed potent myopathy or pravastatin relatively less
toxic than cerivastatin, and measured cell viability using MTT assay .
MTT assay showed significant concentration-dependent decrease of
cell viability by treatment of statins and revealed cerivastatin is much
more toxic than pravastatin . It seems that DNA microarrays could be
very helpful not only to predict drug-induced toxicity, but also to better
understand the mechanism of actions of drug . Using DNA microarrays,
we discovered 522 genes that are specifically responsible to cerivastatin-induced
muscle cell toxicity and these genes correctly classified
as muscle toxicity group. Moreover fifteen genes that are the potential
candidates as myopathy biomarkers were selected . Among them,
seven genes are the sensitive genes that were showed response even
at the low toxic dose, and eight genes are responded in dose-dependent
manner .
P06.276
Genetic association study of Kalirin and Ropporin with ischemic
stroke
T. Krug 1 , H. Manso 1,2 , L. Gouveia 3 , B. V. Fonseca 1 , I. Albergaria 2 , G. Gaspar 2 ,
M. Correia 4 , M. Viana-Baptista 5 , R. M. Simões 6 , A. N. Pinto 6 , R. Taipa 4 , C.
Ferreira 7 , J. R. Fontes 7 , M. R. Silva 8 , J. P. Gabriel 8 , I. Matos 9 , G. Lopes 4 , J. M.
Ferro 3 , A. M. Vicente 1,2 , S. A. Oliveira 1 ;
1 Instituto Gulbenkian de Ciencia, Oeiras, Portugal, 2 Instituto Nacional de Saude
Dr. Ricardo Jorge, Lisboa, Portugal, 3 Hospital de Santa Maria, Lisboa, Portugal,
4 Hospital Geral de Santo Antonio, Porto, Portugal, 5 Hospital Garcia de Orta,
Almada, Portugal, 6 Hospital Fernando Fonseca, Amadora, Portugal, 7 Hospital
Sao Marcos, Braga, Portugal, 8 Hospital de Sao Pedro, Vila Real, Portugal,
9 Hospital Distrital de Mirandela, Mirandela, Portugal.
Introduction: Cerebrovascular and cardiovascular diseases such as
stroke and coronary artery disease (CAD) are the leading causes of
death and disability worldwide . They are complex disorders resulting
from the interplay of genetics and environment, and may share several
susceptibility genes . Recently, an association-mapping study in
the chromosome 3 linkage peak for CAD found that the rs9289231
SNP in the Kalirin gene (KALRN) was associated with CAD in multiple
datasets . KALRN is involved, among others, in the inhibition of inducible
nitric oxide synthase, in guanine exchange factor activity, and in
the Rho GTPase-signaling pathway . The goal of the present study was
to determine whether SNPs or haplotypes in the KALRN gene region,
which includes the Ropporin gene (ROPN1), predispose to ischemic
stroke (IS) in a cohort of Portuguese patients and controls .
Methods & Materials: We genotyped 27 tagging SNPs in the KALRN-
ROPN1 chromosomal region, on 565 IS patients and 518 unrelated
controls, and performed single-marker association tests .
Results: Intronic SNP rs11712619 in KALRN was associated with IS
risk in unadjusted (allelic and genotypic p=0 .003) and adjusted (logadditive
model p=0 .027, adjusted for age-at-examination, hypertension,
diabetes, ever smoking/drinking) tests . A block of six SNPs (from
rs2280422 to rs12637456), located mostly in the ROPN1-KALRN intergenic
region, had a modest (0 .02