2009 Vienna - European Society of Human Genetics

Complex traits and polygenic disorders
P09.036
Lack of association between neprilysin (NEP) gene
polymorphisms and complex Regional Pain syndrome (cRPs)
in a German cohort
K. Huehne 1 , U. Schaal 1 , S. Leis 2 , T. Geisslein 1 , B. Rautenstrauss 3 , F. Birklein 4 ,
C. Maihöfner 5 , A. Winterpacht 1 ;
1 Institute of Human Genetics, Friedrich-Alexander-University, Erlangen, Germany,
2 Department of Neurology, University Hospital, Salzburg, Austria, 3 MGZ,
Medical Genetics Center, Munich, Germany, 4 Department of Neurology, Pain
Research Unit, University of Mainz, Mainz, Germany, 5 Department of Neurology,
University Hospital Erlangen-Nuremberg, Erlangen, Germany.
Complex Regional Pain Syndrome (CRPS) is a chronic neurological
disorder chracterized by disabling pain, swelling and impairment of
motor function. The disorder usually develops after minor trauma or
surgery. Patients with CRPS reveal enhanced sensitivity to experimentally
applied substance P (SP), a neurokinin responsible for neurogenic
inflammation. The extended sensitivity is even present at symtom-less
stages of the disease. A genetic background for the disease is assumed
based on some familial cases and studies describing association
of HLA antigens and CRPS. A functional candidate gene approach
prompted us to determine whether neprilysin (NEP) as a SP degrading
endopeptidase influences disease susceptibility in a German cohort of
patients. DNA was obtained from 325 CRPS patients and 376 controls.
Initially, we demonstrated association of a GT- repeat polymorphism
in the promoter region of NEP with CRPS (p < 0.05). To confirm this
result, 21 SNPs throughout the NEP gene region were genotyped using
allelic discrimination taqman assays. No significant differences in
genotype frequencies were observed between controls and CRPS patients.
In conclusion, our study did not reveal any association between
NEP polymorphisms and CRPS. In addition, our data demonstrate the
limitations of candidate gene association studies relying on single associated
polymorphisms.
P09.037
A high diploid copy number of the beta-defensin cNV associates
with severe cOPD
H. Nuytten1 , D. Lieven2 , K. Nackaerts2 , S. Vermeire2 , J. Cassiman1 , H. Cuppens1
;
1 2 K.U.Leuven, Leuven, Belgium, University Hospital of Leuven, Leuven, Belgium.
Chronic Obstructive Pulmonary Disease (COPD) is characterized by
airflow limitation that is not fully reversible. Smoking is a major environmental
risk factor for developing COPD, but not all smokers develop
COPD. Linkage studies have shown that the chromosomal 8p23 region,
where the defensin repeat region is located, is linked with susceptibility
to airflow obstruction. The beta-defensin CNV is polymorphic
between individuals and therefore the dosage of these defensin
genes/proteins varies.
The aim of this study was to investigate whether the diploid beta-defensin
copy number associates with COPD.
We developed a real time PCR assay to quantify the number of betadefensin
repeats in this region. For this purpose we generated 4 concatemeric
constructs with 1 copy of DEFB1 and a particular number of
DEFB4 copies, which ranged from 1 to 4 copies. Using these controls
as standards, the number of defensin repeats could be accurately determined
in DNA samples.
The diploid beta-defensin copy number was determined in 110 severe
COPD patients, 149 age-matched smoking control individuals, and
121 adult control individuals. The severe COPD group had a mean
diploid beta-defensin copy number of 5.1, the two control populations
had a mean copy number of 4.3. The T-test P-values comparing the
COPD patients with the control populations were 4*10-14 and 8.2*10- 8 respectively. The distributions in the two control populations were
equal (P-value = 0.8). A strong association between the diploid betadefensin
copy number and the diagnosis of severe COPD was thus
found with an Odds ratio of 5 (95% CI 2.57-8.78).
P09.038
Detection and quantification of intestinal mucosa associated
Escherichia coli in crohn`s disease patients by multiplex real
time PcR
J. Šimenc, U. Potočnik;
University of Maribor, Maribor, Slovenia.
Inflammatory bowel diseases encompasses Crohn`s disease and ulcerative
colitis, which are exhibited as chronic intestinal inflammation
of unknown etiology. Resident bacterial flora plays a pivotal role in
the disease development and perpetuation of inflammation. In several
recent studies Escherichia coli has been suggested as a key feature of
Crohn`s disease, with increased concentration in the intestinal mucosa
tissue. Previous studies, based on plate enumeration had implied a
correlation between the amounts of tissue associated Escherichia coli
and a form or phase of the disease. On the other hand, 16S rDNA
gene real-time PCR quantification studies of entire Enterobacteriaceae
family showed contradictory results, with no change or reduction
of enteric bacteria in Crohn`s disease tissue. In the present study, we
applied multiplex PCR for relative quantification of Escherichia coli in
the Crohn`s disease patients and patients with polyps. A region of 16S
rDNA gene was amplified with a single broad range primer pair, targeting
Proteobacteria with two hydrolysis probes; Escherichia coli specific
and universal probe for normalization. Normalized values of Escherchia
coli amount between healthy and inflamed mucosa of Crohn`s
patients were compared and no statistical significant difference was
found (p=0,754). Then amount of Escherichia coli between Crohn`s
disease mucosa and healthy mucosa of polyposis patients was compared
and significant reduction of Escherichia coli was found (p=0,04).
Finally, amount of Escherichia coli between healthy tissue and polyps
from polyposis patients showed significant reduction of Escherichia
coli in polyps as compared with healthy tissue (p=0,032).
P09.039
Analysis of three polymorphisms in TNF-alpha gene: c.-857c>t,
c.420c>t, c.750A>t, among Polish patients with crohn’s
disease.
L. Jakubowska-Burek 1,2 , M. Kucharski 1 , M. Kaczmarek 3 , J. Hoppe-
Gołębiewska 3 , O. Zakerska 3 , K. Linke 1 , M. Drews 4 , R. Słomski 5,6 , R. Marciniak 4 ,
A. Dobrowolska-Zachwieja 1 ;
1 Department of Gastroenterology, Human Nutrition and Internal Diseases, University
School of Medical Sciences, Poznan, Poland, 2 Postgraduate Studium
of Molecular Medicine, Warsaw, Poland, 3 Institut of Human Genetics, Polish
Academy of Sciences, Poznan, Poland, 4 Department of General, Gastroenterological
and Endocrinological Surgery, University School of Medical Sciences,
Poznan, Poland, 5 Department of Biochemistry and Biotechology, University of
Life Sciences, Poznan, Poland, 6 Institut of Human Genetics, Polish Academy of
Sciences, Poznań, Poland.
Crohn’s disease (CD) together with ulcerative colitis (UC) belongs to
inflammatory bowel diseases (IBD). Etiology of the CD is still unknown
but it is suspected that genetic, environmental and immunological factors
play a major role in the background of CD. Currently, the morbidity
in the European Union oscillates around 5 new cases per 100,000
people per 1 year and it is still higher.
During last few years some genes of predisposition to CD were localized,
including the most significant locus - pericentromeric region of
chromosome 16 (16p12-q13), called IBD1 and CARD15/NOD2 gene
in this region. Numerous studies showed that polymorphisms in this
gene are associated with susceptibility to CD. Other studies implicate
regions: 12p13 (IBD2), 6q13 (IBD3), 14q11 (IBD4) or 5q31 (IBD5).
One of the genes investigated in the field of Crohn’s pathogenesis was
also TNF-alpha gene.
The aim of this study was to analyze three SNPs within the TNF-alpha
gene: c.-857C>T, c.420C>T, c.750A>T.
Investigated group consisted of 96 Polish patients with CD and apropriate
population group. Genotyping was performed by pyrosequencing.
We did not observed any variation among Polish patients with CD and
population. We concluded that polymorphisms: c.-857C>T, c.420C>T,
c.750A>T cannot be used as markers of Crohn’s disease susceptibility
in Polish population.
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