2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
Modena, Italy.
Retinitis Pigmentosa (RP) is one of the leading causes of visual handicap
in the world population and is characterized by high genetic heterogeneity
. The study of the disease mechanisms and the development
of efficient therapeutic approaches have mostly relied on the
availability of animal models for this condition, so far . Nevertheless,
little information is available about the RNA expression profiles of RP
genes in the human retina . To overcome this lack of information, we
generated an expression atlas of 34 known RP genes in human and
murine retinas . The atlas can be freely accessed at http://www .tigem .
it/RPexp/ . The vast majority of the genes analyzed displayed similar
patterns between human and mouse retina . We observed different
expression patterns for the CNGB1, USH2A and FSCN2 genes with
respect to previously reported profiles. Additionally, we detected different
expression profiles for the RPGR, CA4, PAP1, RGR and RLBP1
genes in human and mouse retina . The differences observed in the
expression patterns of some genes in human and mouse will open
new perspectives on the function of these genes and on their putative
roles in disease pathogenesis .
P05.170
Overexpression of CERKL, a Retinitis Pigmentosa gene,
protects cells from apoptosis under oxidative stress conditions
A. Garanto 1,2 , M. Tuson 1,3 , R. Gonzàlez-Duarte 1,2 , G. Marfany 1,4 ;
1 Dept. de Genètica; Universitat de Barcelona, Barcelona, Spain, 2 CIBERER.
Instituto de Salud Carlos III, Barcelona, Spain, 3 Sloan-Kettering Institute, New
York, NY, United States, 4 IBUB. Universitat de Barcelona, Barcelona, Spain.
Retinitis Pigmentosa (RP) is a highly heterogeneous genetic disease,
where more than 30 causative genes have been already reported . RP
is characterised by progressive retinal neurodegeneration due to photoreceptor
apoptosis. Our group identified a previously unannotated
gene, CERKL, as responsible for this disease in 3 different spanish
families . CERKL encodes a pressumptive lipid kinase that shares similarity
to the human ceramide kinase . Ceramides are sphingolipids, a
group of membrane lipids that have been increasingly involved in the
regulation of relevant cellular and physiological processes, such as
cell growth, differentiation, apoptosis and inflammation. Therefore, we
hypothesised that CERKL would play a key role in controlling photoreceptor
survival/death . Overexpression of the CERKL enzyme in
transiently transfected cultured cells clearly protects cells from apoptosis
under oxidative stress conditions . This protection starts as early
as 4 hours, but becomes more prominent at 24 hours post-treatment,
where apoptosis is reduced two-fold by CERKL expression . We are
now actively searching for its retinal substrate . Our in vitro and in vivo
preliminary results do not support that ceramide/s are the direct substrate
for this enzyme . Subcellular localisation of CERKL shows that
it is associated to membranes, such as endoplasmic reticulum, Golgi
and Trans-Golgi vesicles, although it can also be found in the nucleus
in some cells, pointing to shifts in localisation depending on the cellular
state . Dissecting the function of CERKL will provide new clues
on the sphingolipid role on photoreceptors and unveil new targets for
therapeutical approaches .
P05.171
mutational analysis of RHO and RDs genes in patients with
autosomal dominant form of retinitis pigmentosa from Volga-
Ural region of Russia
E. R. Grinberg1 , L. U. Dzhemileva1 , I. S. Zaidullin2 , E. K. Khusnutdinova1 ;
1 2 Institute of biochemistry and genetics, Ufa, Russian Federation, Research
Institute of Eye Diseases, Ufa, Russian Federation.
Purpose: To identify mutations in the RHO and RDS genes in patients
with autosomal dominant form of retinitis pigmentosa (adRP) from Volga-Ural
region of Russian Federation . Retinitis pigmentosa, the most
common hereditary cause of blindness, comprises a clinically and genetically
heterogeneous group of retinal disorders . It affects about one
in 5 000 individuals worldwide
Methods: 37 patients with adRP confirmed by pedigrees were examined
clinically and with visual function tests . The 5 exons of RHO
and 3 exons of RDS genes were analyzed for sequence changes by
single-strand conformation polymorphism analysis (SSCP) and further
sequencing .
Results: We detected known mutation Pro347Leu, novel mutation Arg-
252Pro and two polymorphisms IVS1+10g>a, IVS3+4c>t in RHO gene .
There were statistically significant differences in allele and genotype
frequencies of sequence change IVS3+4c>t of RHO gene in affected
patients with adRP and in controls . According to our data, this polymorphism
might be pathogenic . Recently were reported 16 possible
combinations of the exon 3 RDS gene four SNPs and detected four
from 16 possible combinations (minihaplotypes): G 1147 A 1166 G 1250 C 1291
(I), C 1147 A 1166 A 1250 C 1291 (II), C 1147 G 1166 A 1250 C 1291 (III) and G 1147 A 1166 G 1250 T 1291
(IV) . Sequencing results for variant positions in exon 3 RDS gene in
adRP patients from Volga-Ural region revealed all four minihaplotypes
described above and minihaplotype C 1147 A 1166 A 1250 T 1291 (V) . Minihaplotype
C 1147 A 1166 A 1250 C 1291 (II) was revealed only in patients and it might
be linked with RP .
Conclusions: To optimize the DNA diagnostics of retinitis pigmentosa,
it is necessary to analyze patients from various ethnic groups . Our
study helps in molecular characterization of RP in Russia .
P05.172
Knock-down of Cox5a in HEK293 cells
D. Fornuskova, L. Stiburek, J. Zeman;
Faculy of Medicine, Prague 2, Czech Republic.
Cox5a is one of 13 structural subunits of cytochrome c oxidase (COX),
the terminal enzyme of respiratory chain . We used RNA interference
(RNAi) to down-regulate steady-state level of Cox5a subunit and analyzed
impact of its knock-down on COX assembly .
We constructed seven derivatives of pCMV-GIN-ZEO plasmid coding
for hairpins aimed at different positions of COX5A mRNA . A Cox5a protein
has a long half-life (a level of the protein is 48 hours post-nucleofection
unchanged using immunoblotting) . Therefore we introduced
COX5A coding sequence into the maxFP-Red - N plasmid to encode
fusion protein . The marker plasmid was co-transfected with RNAimediating
plasmid derivatives (the higher the level of fusion protein
the higher the leakage of individual hairpin-mediated RISC systems) .
A fluorescence of fusion protein was found rapidly lower compared
to maxFPRed marker thereby complicating a setting of FACS measurements,
but Western Blot of fusion protein with COX5A antibody
gave an acceptable result . To optimize the detection of RNAi-potential
through fluorescence by FACS, we re-cloned COX5A coding sequence
into 3´UTR of maxFPRed marker. The final transcript contains
target sequence for RNAi but leads to translation of merely maxFPRed
marker . Fluorescence intensities were comparable with that obtained
at empty plasmid .
Based on the above-mentioned methods, we chose three candidate
shRNAs and prepared stable cell lines, where depletion of Cox5a was
confirmed using SDS immunoblotting. Also specific activity of COX
was revealed decreased . BN-PAGE showed diminished level of COX
holoenzyme and its assembly intermediates .
Supported by GACR 305/08/H037 and GAUK 1/2006/R .
P05.173
the challenges encountered in diagnostic screening of the
RYR1 gene
S. M. Lillis1 , T. Lepre1 , H. Zhou2 , F. Muntoni2 , H. Jungbluth1 , S. Abbs1 ;
1 2 Guy’s & St Thomas’ NHS Foundation Trust, London, United Kingdom, Neuromuscular
Unit, Hammersmith Hospital, London, United Kingdom.
Mutations in the skeletal muscle ryanodine receptor gene (RYR1) are
implicated in a number of congenital myopathy phenotypes including
central core disease, multi-minicore disease and centronuclear myopathy
.
We have developed a high throughput 384-well sequencing approach
for screening the 106 exons of RYR1 . This method uses tagged primers
and robotics to overcome the practical challenges faced by screening
such a large gene . To date we have tested over 44 patients and
here we report on the complexities which have arisen .
Certain RYR1 related disorders may exhibit both autosomal dominant
and recessive modes of inheritance; it is not always certain whether we
are searching for one or two mutations. The identification of missense
changes of unknown pathological significance complicates this.
Our mutation screening is further complicated due to the marked phenotypic
variability: mutations in RYR1 can manifest in a wide range
of clinical phenotypes whilst similar phenotypes may result from mutations
in a number of different genes excluding RYR1 . Additionally
there are reports of RYR1 mutations that only exhibit symptoms on a
mono-allelic background .