2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
rithms. Using this integration, the number of potential targets predicted
using bioinformatic tools was significantly reduced. These targets are
now being validated to determine whether they are true biological targets.
Research supported by the European Commission FP6 Integrated
Project EuroHear.
P11.033
Ribosomal protein S19 and S24 insufficiency in Diamond-
Blackfan anemia cause prolonged cell cycles with distinct
arrests in non-hematopoietic cells
J. Badhai, A. Fröjmark, E. Davey, J. Schuster, N. Dahl;
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University,
Uppsala, Sweden.
Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized
by a specific decrease of erythroid precursor cells. Although
anemia is the most prominent feature, the disease is also associated
with growth retardation and congenital malformations in 50% of patients.
Heterozygous mutations in one of the seven ribosomal protein
(RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RP-
L35a have been identified in approximately 35% of patients. We established
primary fibroblast cell lines from DBA patients with truncating
mutations in the RPS19 (c.72-2A>C) and RPS24 (c.1A>G) genes. A
growth assay showed that fibroblasts from DBA patients with truncating
mutations have a marked reduction in proliferative capacity. Mutant
fibroblasts are associated with cell cycles extended by 50% and 33%,
respectively, when compared to w.t. cells. RPS19 mutant fibroblasts
accumulate in the G1 phase suggesting a G1/G0 arrest, whereas
RPS24 mutant cells are significantly reduced in the G2 phase. We also
observe a concomitant down regulation of the small subunit proteins
in mutant cells. The mutations result in impaired rRNA maturation and
ribosomal subunit assembly. The results show that the major cause
of impaired growth in RPS19 and RPS24 insufficient fibroblasts is a
delayed cell cycle with distinct profiles. We suggest that the reduced
proliferative capacity is an important contributing mechanism behind
extra-hematological features in DBA.
P11.034
comparison of DNA methylation patterns in three mouse tissue
types.
B. F. Johnson1 , C. J. Davidson1 , M. Kondo2 , L. K. Joe1 , S. R. Berosik1 , A.
Chhibber1 , R. N. Fish1 , S. C. Hung1 , J. Lee1 , R. A. Padilla1 , D. Rodriguez1 , A. A.
Pradhan1 , A. C. Felton1 ;
1 2 Life Technologies, Foster City, CA, United States, Applied Biosystems-Japan,
Tokyo, Japan.
Enzymatic methylation of the cytosine residues in genomic DNA
(gDNA) has been shown to correlate with gene expression. Methylation
of cytosines (C) at CpG motifs, usually in the promoter regions of
genes, will shut down expression of the gene in complex biological processes.
Technologies like microarray and next generation sequencing
allow identification of methylation patterns on a genome-wide scale but
capillary electrophoresis analysis is ultimately used for detailed information
of each CpG in the amplicon and remains the gold standard in
validating DNA methylation results. In this study bisulfite treated gDNA
of a specific gene region from three mouse tissues was cloned into
pGEM-T vector, amplified and sequenced with BigDye® Terminator
Cycle Sequencing v3.1 chemistry, and analyzed by capillary electrophoresis.
A number of factors affect the reliability of methylation patterns
revealed by sequencing including the purity of the gDNA, the
efficiency of bisulfite conversion, and the interaction of modified DNA
with primers and DNA polymerase. The extent of cytosine conversion
by bisulfite is affected by the presence of protein associated with gDNA
, the denaturation state of the target DNA and well as the quantity of
DNA in the conversion reaction. In this study a control using methylated
and unmethylated reference DNA is described that validates the
bisulfite conversion conditions for improved reliability of the resultant
methylation profile.
P11.035
sequence capture Approaches coupled to Next Generation
sequencing to identify candidate mutations causing inherited
Disorders
L. Dannenberg 1 , T. Albert 1 , D. Burgess 1 , J. Jeddeloh 1 , M. Bainbridge 2 , M.
D’Ascenzo 1 , D. Muzny 2 , L. Nazareth 2 , X. Zhang 1 , R. Gibbs 2 , V. Ott 1 ;
1 Roche NimbleGen, Inc., Madison, WI, United States, 2 Human Genome Sequencing
Center, Baylor College of Medicine, Houston, TX, United States.
We have developed an optimized oligonucleotide microarray for genomic
selection and targeted sequencing of the entire human CCDS
database. The NimbleGen Sequence Capture 2.1M Human Exome
array contains 2.1 million capture oligonucleotides targeting ~180,000
exons, totaling ~34 Mb of sequence. We have used this array, coupled
to 454 Titanium sequencing technology on the Genome Sequencer
FLX instrument, to sequence the exons of patients with idiopathic ataxia
to ~10X average coverage. Data analysis has revealed previously
uncharacterized mutations in genes known to cause neurological disorders.
This approach has the potential to replace time-consuming and
laborious classical genetic methods, and may lead directly to functional
candidate mutations for highly penetrant inherited phenotypes.
P11.036
comparative analysis of expression levels of genes of the
extracellular matrix proteins from human normal and scar skin
fibroblasts
A. Solovyeva1 , M. Khotin2 , L. Turoverova2 , N. Yudintseva2 , M. Blinova2 , G.
Pinaev2 , D. Tentler2 ;
1 2 Saint-Petersburg State University, Saint-Petersburg, Russian Federation, Institute
of Cytology RAS, Saint-Petersburg, Russian Federation.
A major reason for scar formation is an abnormality in the synthetic
activity of fibroblasts, which are the major producers of the extra cellular
matrix (ECM) in different types of tissues. Compositional ratios of
structural ECM proteins are strongly tissue-specific and variations in
these protein compounds may contribute to architectural and functional
heterogeneity. A particular kind of ECM organization is the epidermal
basal membrane (BM). The main structural proteins of BM are laminin,
collagen and fibronectin. In order to reveal the possible reasons
responsible for scar skin formation, we compared the expression of
genes encoding main proteins of the BM between normal, embryonic
and scar skin human fibroblasts in culture. Using semi-quantitative RT-
PCR method, we have estimated the expression levels of the following
genes: LAMA2, LAMB1, LAMB2, LAMY1 encoding subunits of laminin;
COLIA1 and COLIA2 encoding subunits of collagen type 1; and COLI-
VA1, COLIVA2, COLIVA4 and COLIVA5 encoding subunits of collagen
type 4. The results showed that expression levels of genes LAMA2,
LAMY1, COLIVA4 and COLIVA5 depended on type of skin fibroblasts
origin. The expression levels of COLIVA4, LAMA2 and LAMY1 genes
were higher in normal skin fibroblasts and the expression levels of
COLIVA5 were lower in scar skin fibroblasts comparing to two other
types of fibroblasts. No other correlations were revealed. We have also
developed a sparing method of ECM proteins isolation from cultured
fibroblasts. Our next goal is to estimate whether differences identified
in genes expression on RNA levels also reflect different amounts of the
corresponding protein products.
Acknowledgements: This work was supported by program MCB of
Russian Academy of Sciences
P11.037
A molecular combing approach for FsHD: direct visualization of
the FsHD locus in individual DNA molecules for straightforward
diagnosis and genetic and epigenetic explorations
P. Walrafen 1 , K. Nguyen 2,3 , A. Vannier 1 , E. Renard 1 , C. Vovan 2 , C. Chaix 1 , R.
Bernard 2,3 , A. Bensimon 1 , N. Lévy 2,3 ;
1 Genomic Vision, Paris, France, 2 Département de Génétique Médicale - Hôpital
d’enfants La Timone, Marseille, France, 3 INSERM UMR S910 «Génétique et
génomique fonctionnelle», Université de la Méditérranée, Marseille, France.
Facioscapulohumeral dystrophy (FSHD) is the third most common
muscular dystrophy, with autosomal dominant transmission. FHSD is
associated to the contraction of a repeat array at the subtelomere of
chromosome 4q (4q35), which comprises 1-150 copies of a 3.3 kbp repeat
unit, D4Z4. A virtually identical array is present at the subtelomere
of chromosome 10q. FSHD alleles carry 1-10 copies on a 4qA chromosome,
one of two equally frequent variants of the 4qter subtelomere.