2009 Vienna - European Society of Human Genetics

Cancer genetics
P06.089
molecular genetic investigation in patients with lung cancer:
parallel analysis of STRs in cell-free DNA and SNP polymorfisms
within 15q24-q25 region in genomic DNA
E. Hirmerová 1 , A. Panczak 1 , V. Kebrdlová 1 , A. Hořínek 1 , J. Homolka 2 , J.
Štekrová 1 , M. Kohoutová 1 ;
1 Institute of Biology and Medical Genetics, 1st Faculty of Medicine, Charles
University, and General Teaching Hospital, Prague, Czech Republic, 2 1st Department
of Tuberculosis and Respiratory Diseases, 1st Faculty of Medicine,
Charles University, and General Teaching Hospital, Prague, Czech Republic.
Short tandem repeats (STRs) located in/at TP53 (pentaTP53, diTP53),
APC (D5S346, D5S318, D5S299, D5S82), FHIT (D3S1300) and VHL
genes (D3S1560) were analyzed in plasma cell-free DNA (cfDNA),
and, in parallel in genomic DNA, four single-nucleotide polymorphisms
(SNPs) mapping to the region of nicotinic acetylcholine receptor subunit
genes on 15q24-q25. Study was conducted to identify risk genetic
factors in patients with NSCLC (group P), compared to individuals
with non-tumor pulmonary diseases (group C), and anonymized blood
samples from routine laboratory (group A). We have studied loss of
heterozygosity (LOH) in total cfDNA amplified by time-release PCR
with primers for STRs; genomic DNA of the same patient served us
as a control. Further, we have introduced snapshot analysis for SNPs
in genes: LOC123688 (rs931794, rs8034191), CHRNA3 (rs1051730)
and CHRNA5 (rs16969968), recently shown to be associated with risk
for lung cancer. We found out simultaneous presence of LOH in multiple
STR loci in group P in contrast to both control groups, this could
predicate lung cancer, e.g. in differential diagnostics. The highest sensitivity
was shown in microsatellite marker D5S82. In addition, there
were three lung cancer patients where one of two allele of D5S82 or
D5S318 completely disappeared. The analysis of SNPs demonstrated
they form haplotypes except in group P, where 6.7 % of chromosomes
were “recombinant”. We followed the incidence of risky alleles of four
SNPs in all three groups, and compared the structure of data from
both tests.
Supported by the grant project MŠMT CR MSM0021620808
P06.090
mutations of succinate Dehydrogenase and Fumarate
Hydratase. A tcA cycle Gene mutation Database Update
J. Bayley 1 , P. Devilee 1 , P. E. M. Taschner 1 , V. Launonen 2 , I. P. M. Tomlinson 3 ;
1 Leiden University Medical Center, Leiden, The Netherlands, 2 University of
Helsinki, Helsinki, Finland, 3 Cancer Research UK, London, United Kingdom.
Two enzymes of the tricarboxcylic acid (TCA) cycle, fumarate hydratase
(FH) and succinate dehydrogenase (SDH), involved in fundamental
processes of energy production, are now known to be tumor
suppressors.
While deficiencies of FH and SDH(A) result in severe early-onset encephalopathy,
germline mutations of SDH genes are a major cause of
hereditary paraganglioma and pheochromocytoma, and FH mutations
result in the hereditary leiomyomatosis and renal cell cancer (HLRCC)
tumor syndrome.
The SDH mutation database (DB) was launched in 2005 and with the
inclusion of FH is now the TCA Cycle Gene Mutation Database. The
TCAC DB is gene-centered and based on LOVD, with each gene having
a separate summary page listing general information and providing
access to tables containing variant information and various search
options. The HGVS conform nomenclature provides an accessible resource
easing the description of new mutations.
371 unique variants are currently described in the database. We describe
the current status of each gene and recent developments.
In 2008 the database attracted >75,000 page hits, a doubling compared
to 2007, and saw an average monthly use by 377 unique IP
addresses. The increasing community use of the TCAC DB indicates
widespread acceptance and increasing utility.
The TCAC DB represents a valuable resource for clinicians, clinical
geneticists, and researchers interested in paraganglioma/ pheochromocytoma
and HLRCC.
P06.091
Anti-mUc1 VHH can redirect chimeric antigen receptor (cAR)
cytotoxic effector function
S. Aghaee Bakhtiari, F. Rahbarizadeh, F. Jafari Iri-Sofla, M. Rasaee;
Tarbiat Modares University, Medical Biotechnology department, Tehran, Islamic
Republic of Iran.
Chimeric antigen T cell receptors provide a good approach for adoptive
immunotherapy of cancer, especially in the context of cancerous
cells that fail to express major histocompatibility complex antigen and
co-stimulatory molecules. Clinical applications of these receptors are
limited, mostly due to xenogenic origin of the antibodies which cause
immunogenic reactions. VHH are the smallest fragments of antibodies
that have great homology to human VH and low immunogenic potential.
MUC1 is a highly attractive immunotherapeutic target owing
to increased expression, altered glycosylation, and loss of polarity in
more than 80% of human malignancies. We used anti-MUC1 VHH as
an antigen binding domain, CD28 and CD3ζ as signaling domains and
IgG3 as a spacer in a chimeric receptor construct. This construct was
transfected to Jurkat cells. The transfected Jurkat cells were exposed
to MUC1 positive MCF7 cells. Then we analyzed the secretion of IL2,
proliferation of Jurkat cells and death of MCF7 cells. These data revealed
that the VHH chimeric receptor can target tumor associated
antigen positive cells. Regarding the efficient and specific function of
VHH chimeric receptor and non immunogenic nature of VHH, these
chimeric receptors might be used as promising candidates for clinical
applications.
P06.092
molecular Analysis of t-cell Receptor Gene Rearrangements
L. K. Joe1 , S. R. Berosik1 , A. Chhibber1 , C. J. Davidson1 , R. N. Fish1 , S. Hung1 ,
B. F. Johnson1 , J. Lee1 , R. A. Padilla1 , D. Rodriguez1 , A. Sartori1 , M. Yamazaki2 ,
A. A. Pradhan1 , A. C. Felton1 ;
1 2 Life Technologies Corporation, Foster City, CA, United States, Hitachi High
Technologies, Naka, Japan.
The evaluation of clonality on the T cell receptor locus can be an important
tool in detecting T-cell non-Hodgkin lympoproliferations. The
locus is comprised of a 160 kb region on Chromosome 7 and has
well defined variable (V), joining (J) and constant (C) gene segments.
Molecular interrogation of the rearrangements in the variable and joining
segments can be used to compare fragment sizes of DNA from
a normal polyclonal T-cell population (many different sizes) to DNA
from a clonal T cell population with suspected lymphoproliferations
(few different sizes). The comparison can be accomplished with PCR
and subsequent fragment size analysis via capillary electrophoresis.
PCR methods using fluorescent labeled primers designed to anneal
to the flanking, conserved, regions of interest on the locus have been
established. We describe new methods used to accomplish the DNA
comparison using Capillary Electrophoresis (CE) systems that produce
consistent results. Use of these CE systems could greatly benefit
an individual or association of researchers with multiple instruments in
many locations. Traditionally, conclusions were drawn by comparing
the DNA fragments by visual inspection. We demonstrate new software
analysis methods that allow the scientist to make quantitative
conclusions.
P06.093
Presence of activating KRAS mutations correlates significantly
with expression of tumour suppressor genes DcN and tPm1 in
colorectal cancer
V. Mlakar 1 , G. Berginc 1 , Z. Štor 2 , M. Rems 3 , D. Glavač 1 ;
1 Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine,
Ljubljana, Slovenia, 2 Department of Abdominal Surgery, University Medical
Centre Ljubljana, Ljubljana, Slovenia, 3 Department of Surgery, Jesenice Hospital,
Jesenice, Slovenia.
Despite the identification of the major genes and pathways involved in
the development of colorectal cancer (CRC), it has become obvious
that several steps in these pathways might be bypassed by other as
yet unknown genetic events that lead towards CRC. To improve our
understanding of the genetic mechanisms of CRC development, we
used microarrays to identify novel genes involved in the development
of CRC. Using real-time PCR, we also searched for chromosomal
abnormalities within candidate genes and the expression pattern in
the case of KRAS mutation. We detected significant previously unde-