2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
variants present and proved the sensitivity of the method for mutation
detection. In addition, we showed that we could exploit the multiplexed
PCR amplicons to determine individual Copy Number Variation (CNV),
increasing the spectrum of detected variations to both genetic and genomic
variants. We conclude that our straightforward procedure substantially
expands the applicability of the massive parallel sequencers
for sequencing projects of a moderate number of amplicons (50
- 500) with typical applications in resequencing exons in positional or
functional candidate regions and molecular genetic diagnostics. Completely
in line with this conclusion, we are now developing assays for
the CFTR and BRCA1/BRCA2 coding sequences.
P11.002
Promoter polymorphism -368 c/t of the acetyl-coA carboxylase
2 gene ACACB influences activity and nuclear protein binding in
HepG2 cells
A. K. Lee, T. Kyriakou, S. D. O’Dell;
Nutritional Science Division, London, United Kingdom.
Acetyl-CoA carboxylase 2 (ACC2) catalyses the formation of malonyl-
CoA, a key regulator of fatty acid oxidation. Increased ACC2 activity
would reduce fatty acid oxidation because malonyl-CoA inhibits entry
into mitochondria via carnitine palmitoyltransferase I (CPTI). The transcriptional
regulation of the ACC2 gene (ACACB) is complex and involves
many factors which may be tissue-specific. In liver, it has been
established that promoter P-II controls transcription of the ACACB
gene and sterol regulatory element-binding protein-1 (SREBP-1)
regulates expression. We studied the functional impact of a single
nucleotide polymorphism (SNP) in the promoter region -368 C/T
(rs16939972) in HepG2 cells in the presence of SREBP-1a. The -368
C/T SNP was selected as the closest to known SRE sites, validated
on dbSNP and occurring at a frequency of over 0.05 in the CEPH
population (MAF=0.25). The promoter construct carrying the -368 T
allele showed 1.35 fold lower activity than the construct carrying the
-368 C allele. Electrophoretic mobility shift assays (EMSAs) revealed
the -368 T allele has a higher affinity for nuclear proteins than the -368
C allele, suggesting the -368 C/T SNP may bind to a repressor. EMSA
competition experiments using unlabelled oligonucleotides containing
transcription factor binding motifs, showed that the -368 C/T SNP may
bind to C-Myb, GATA and/or glucocorticoid receptor (GR) proteins. In
conclusion, the data suggest the ACACB -368 C/T polymorphism is
a regulatory SNP that affects promoter activity in HepG2 cells in the
presence of SREBP-1a and alters nuclear protein binding affinity.
P11.003
molecular and cytogenetic characterisation of human albumin
transgenic goat fibroblasts as a source of nuclei in the somatic
cloning
A. Wozniak 1 , D. Lipinski 1,2 , A. Nowak 2 , J. Zeyland 2 , K. Nuc 2 , B. Rynska 3 , R.
Slomski 1,2 ;
1 Institute of Human Genetics Polish Academy of Sciences, Poznan, Poland,
2 University of Life Sciences, Department of Biochemistry and Biotechnology,
Poznan, Poland, 3 3National Research Institute of Animal Production, Department
of Animal Reproduction Biotechnology, Balice, Poland.
The production of pharmaceutically important human proteins in the
mammary gland of transgenic animals constitutes an important field
of biotechnology.
In this study goats’ fibroblasts were transfected by lipofection with
transgene, which contained human gene encoding albumin under the
control of the tissue specific WAP promoter. Transfected fibroblasts
were cultured with selective medium with blasticidine.
Transgene integration was examined by PCR method. Chromosomal
aberrations were examined using the GTG-binding pattern. Fluorescence
in situ hybridization (FISH) enabled the mapping of transgene
specific DNA sequences.
Transgenic cells are going to be used as a source of nuclei in the experiments
of obtaining transgenic goats by somatic cloning technique.
The application of tissue specific WAP promoter allows to reduce the
expression of the transgene to mammary gland. Human albumin is
going to be found only in the milk of animals being accurately in the period
of lactation. After the separation of albumin from milk components
it would be applied in medical treatment.
P11.004
Rare Allele Enrichment by snapback Primer
L. Zhou1 , D. Smith2 , C. Wittwer1,3 ;
1Department of Pathology, University of Utah School of Medicine, Salt lake
City, UT, United States, 2ARUP Laboratories, Salt lake City, UT, United States,
3ARUP Laboratories, Salt Lake City, UT, United States.
Allele specific PCR for selective amplification of minority alleles is
widely employed in detecting cancer mutations. Allele specific PCR
using Snapback primers is a closed-tube intramolecular method that
does not require fluorescently labeled probes. Only two PCR primers
are required, with the addition of a short-tail of nucleotides on one
primer that results in an intramolecular hybridization probe. Genotyping
is performed by high resolution melting of the hairpin. Rapid cycle
PCR enables selective amplification of minority alleles by lowering the
extension time to 0 second. Using a carousel LightCycler® or LS-32
(Idaho Technology), this requires less than 25 minutes. High-resolution
melting is then performed on an HR-1 or LS-32. The detection
sensitivity of point variants is up to 1 in 1000. A single hotspot mutation
of the BRAF gene (1799 T>A, V600E) was analyzed in thyroid
tumor tissue and needle samples from 45 genotyped patients. It is
the most common change in papillary thyroid carcinoma resulting
in more than 80% of thyroid cancers. The sensitivity and specificity
were 100% compared to histology and other molecular methods. An
EGFR exon19 in-frame deletion was used for small deletion detection;
a 1:10000 deletion mutations to wild type ratio could be analyzed. Somatically
acquired mutations in the EGFR gene in non-small cell lung
cancer are associated with a significant clinical response to tyrosine
kinase inhibitors. Enriching minority alleles with Snapback primers is
attractive because only PCR reagents and dsDNA dye are needed. No
expensive modified oligonuleotides, separations, purification or addition
steps are necessary.
P11.005
impaired activity of serum alpha 1-antitrypsin in carriers of
p.G320R variant
M. Ljujic 1 , A. Topic 2 , A. Nikolic 1 , A. Divac 1 , M. Grujic 3 , M. Mitic-Milikic 3 , D. Radojkovic
1 ;
1 Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia,
2 Institute of Medical Biochemistry, Faculty of Pharmacy, Belgrade University,
Belgrade, 3 Institute for Lung Disease and TB, University Clinical Center of Serbia,
Belgrade, Serbia.
The alpha 1-antitrypsin (A1AT) gene is highly polymorphic, with more
than 100 genetic variants identified so far. Some of these variants can
affect A1AT protein concentration and/or function and lead to pulmonary
and/or liver disease. This study reports on the characterization of
p.G320R variant found in two patients - one with emphysema and other
with lung cancer. This variant results from a single base-pair substitution
in exon 4 of A1AT gene (Gly-320[GGG]→Arg-320[AGG]) and has
been characterized as P by isoelectric focusing. Functional evaluation
of A1AT p.G320R variant was performed by determination of specific
trypsin inhibitory activity in two patients with pulmonary disorders, carriers
of p.G320R variant, and 19 healthy individuals, carriers of normal
A1AT M variants (M1, M2, M3, M1M2 and M1M3). Results showed
that specific trypsin inhibitory activity was lower in both emphysema
and lung cancer patients (2.45 mU/g and 2.07 mU/g respectively) in
comparison with values obtained in carriers of normal A1AT M variants
(range 2.51 - 3.71 mU/g). This A1AT variant is associated with reduced
functional activity of A1AT protein. Considering that it was found in
patients with severe pulmonary disorders, this variant might be of clinical
significance. In order to completely characterize this variant and
estimate whether it is associated with pulmonary disorders, expression
concentration, secretion rate and tendency to aggregate should also
be analyzed.
P11.006
the hunt for de novo chromosomal aberrations in patients with
mR/mcA
K. Kok, G. van der Vries, Y. Swart, H. Alkema, H. Zorgdrager, T. Dijkhuizen, C.
van Ravenswaaij-Arts, B. Sikkema-Raddatz;
UMCG, Groningen, The Netherlands.
Array-based comparative genomic hybridization has become an indispensable
tool in the hunt for small de novo chromosomal aberrations
that are presumed to be present in patients with multiple congenital