2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
P11.090
methylation changes in p53 pathway in non-small cell lung
cancer and their potential association with tP53 mutations
K. Kirotar 1 , R. Kolde 2 , I. Bure 3 , M. Solovjova 1 , T. Vooder 1,4 , K. Välk 1,5 , A. Metspalu
1,6 , N. Tõnisson 1,4 ;
1 Institute of Molecular and Cell Biology / Estonian Biocentre, Tartu, Estonia,
2 Institute of Computer Sciences, Tartu, Estonia, 3 Voronezh State University, Voronezh,
Russian Federation, 4 Tartu University Hospital, Tartu, Estonia, 5 Asper
Biotech, Tartu, Estonia, 6 Estonian Genome Project, Tartu, Estonia.
Methylation changes are common and relatively stable in various types
of cancer. Mutations in TP53 may be present in up to 50% of non-small
cell lung cancer (NSCLC) cases. It is known, that virtually all naturally
occurring mutations in TP53 gene reduce the transactivating ability of
p53 protein.
We have studied methylation of transcriptional start sites of wild-type
p53 target genes in six NSCLC cell lines, two (A549, H292) with wildtype
TP53 gene and four (H23, H520, H1703, H1299) with different
TP53 mutations using combined bisulfite-restriction analysis. Six
normal lung control samples were analyzed. For finding associations
between methylation and expression repression, we carried out 5-aza-
2’-deoxycytidine treatment. The analysis revealed presence of methylation
in nine genes, but all the cell lines had a somewhat different set
of methylated genes.
The number of hypermethylated genes was higher in cell lines with
TP53 mutations. Methylation patterns of p53 transcriptional target
genes were compared to the corresponding expression profiles. Hierarchical
cluster analysis of the methylated genes showed their correlation
with TP53 mutation status. Normal lung tissue controls with
no TP53 mutations found by sequencing, clustered together with the
wild-type TP53 gene cell lines.
5-aza-2’-deoxycytidine treatment caused decrease in the methylation
of several analyzed genes and also induced a mild degree of apoptosis
in the NSCLC cell lines with TP53 mutations.
P11.091
SERPINA1 identifies papillary thyroid carcinoma across four
distinct microarray studies and in independent validation data
K. Vierlinger 1 , M. Lauss 1 , C. Nöhammer 1 , F. Leisch 2 ;
1 ARCS, Seibersdorf, Austria, 2 University of Munich, Munich, Germany.
Background: Several DNA microarray based expression classifiers
for the different clinically relevant thyroid tumor entities have been
described over the past few years. However, reproducibility of these
markers is generally low, mainly due to study biases, small sample
sizes and the highly multivariate nature of microarrays.
Methods: Therefore we adopted a meta analysis approach for four
publicly available microarray datasets on thyroid carcinoma. Data integration
to remove study specific bias was done using Distance Weighted
Discrimination (DWD) and for feature selection and classification
a nearest shrunken centroid algorithm (PAM - Prediction Analysis for
Microarray) was used. Results were validated using independent Microarray
and RTPCR datasets.
Results: Meta-analysis identified a one-gene classifier (SERPINA1) for
papillary thyroid carcinoma. In Meta-analysis, identification of papillary
thyroid nodules versus benign thyroid was achieved with 99% accuracy
(97.9% weighted average accuracy in study crossvalidation). In the
independent microarray validation data, which consisted of all major
thyroid tumor entities, SERPINA1 correctly classifies 100% of nodules
into one of the two classes PTC and non-PTC.
Conclusions: These results show that new insights can be gained using
existing data and indicate a huge potential for future diagnostic
applications.
P11.092
High throughput phenotypic screens in Parkinson‘s Disease
S. Jain 1 , D. Sondervan 1 , P. Heutink 1,2 ;
1 CNCR, Amsterdam, The Netherlands, 2 VUMC, Amsterdam, The Netherlands.
Parkinson (PD) is a neurodegenerative disease with a huge socioeconomic
burden. In PD years of slowly progressing neurodegeneration
set the stage for devastating clinical phases. Current treatments
are only symptomatic which ultimately result in debilitating side effects
thus there is an urgent need to develop therapeutics which stop
and reverse disease progression. 13 loci have been implicated in the
pathogenesis of PD, with only 7 of the genes underlying the loci hav-
ing been identified. These genes have suggested that disruption of a
myriad of molecular pathways (e.g. ubiquitin and proteasome, mitochondrial
function and protein misfolding) can lead to the degeneration
of the substantia nigra. However it remains unclear how mutation of
these genes and disruption of these pathways lead to PD.
To address these issues, we have developed a high throughput and
high content screening facility using siRNA to modulate the expression
of every gene in the genome and determine their effect in a range of
cellular assays related to PD. Several assays have been established
to measure several processes which have been implicated in PD
pathogenesis such as α-synuclein aggregation and phosphorylation,
DJ-1 related functions (apoptosis, translocation and oxidation). The
goal is to construct a detailed molecular pathway of the proteins that
are involved in the function of genes mutated in PD. This will provide a
greater understanding of the molecular pathways involved in PD and
if any of these can be modulated or exploited for their therapeutic potential.
P11.093
Putative interactors of Pax6 indicate novel molecular cascade of
its function
R. Mishra, R. Tripathi, K. Shubham;
Department of Zoology, Varanasi, India.
Objective: The Pax6 (a transcriptional regulator) is critical for brain and
eyes development. The mutation in Pax6 leads to severe brain and
eyes anomalies. Phenotypes are of variable peneterance and expressivity.
It is presumed to be influenced by protein-protein interaction
(matricellular proteins/TGIF/TGF/Neurotrophins)
Methodology: Models of Pax6 interacting proteins were analysed
through on-line servers (STRING and PIP). The interacting proteins
of Pax6 were also explored through co-immunoprecipitation and colocalzation
Significant Results: Novel interaction of Pax6 with SPARC (secreted
protein, acidic and rich in cysteine, a matricellular glycoprotein of
43kDa) was observed. The immunoreactive bands with Ras and p53
were also detected in the sample of brain immunoprecipitated with
anti-Pax6
Conclusions: The observations elucidate novel interactors in the cascade
or hierarchy of Pax6 or SPARC functions. The putative interactors
also provide link to Akt and TGF-beta pathways through SPARC,
Ras and p53. The preliminary observations are useful in exploring molecular
basis of nervous system function
P11.094
Rapid single-molecule haplotyping in patients with sickle cell
disease
S. Menzel1 , J. Qin2 , N. Vasavda1 , S. L. Thein1 , R. Ramakrishnan2 ;
1 2 King’s College London School of Medicine, London, United Kingdom, Fluidigm
Corporation, South San Francisco, CA, United States.
Genome-wide association studies have provided many new genes
influencing disease risk and severity as well as biometric traits. Identifying
causal variants at these new loci has become a bottleneck and
poses a challenge for technology: how to extract useful information
from a focused genomic region in large numbers of samples. Highdensity
SNP genotyping provides a point-by-point survey of the region
of association, but additional information could be gained from the
sequence of SNP alleles on each parental chromosome (phase), i.e.
haplotypes present in each study subject.
We have used an integrated fluidic circuit (IFC) system from Fluidigm
Corporation (the digital array) to analyze multiple SNP loci from individual
molecules of sample DNA and to rapidly obtain haplotype information
over several kb of target sequence. Using this approach, we
were able to phase double heterozygous SNP genotypes over distances
of 0.7, 1.2, and 5.2 kb. We have studied three SNPs (rs9399137,
rs9402685 and rs11759553) at a locus on chromosome 6q23 (HBS1L-
MYB) that modifies clinical severity in sickle cell disease, a monogenic
blood disorder due to a mutation of the β hemoglobin chain. We detected
four common haplotypes (with 46%, 21%, 14% and 13% frequency)
and a single infrequent one (5.5%), which is associated with
milder disease. We plan to extend phasing to a fragment of 24 kb to
help identifying the causative DNA variants at this locus.
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