Poster group A - SYSTEMS MEDICINE / ENVIRONMENT
A.1 - ISThMe: Illustrating Medical Strategies through Multi-agent systems - Casestudy: hypertension
N. Abchiche-Mimouni1 and F. Gueyffier2
1
EA 4526 - Laboratoire d'informatique et de Biologie integrative Universite d'Evry UMR 5558 - Laboratoire de Biometrie et Biologie Evolutive
Facult RTH Laennec - LYON CEDEX 08
2
This paper presents a multi-agent plateform (ISThMe) for the illustration of therapeutic strategies and
for helping prescribers to personalize the treatment in the case of hypertension. ISThMe has been used
to compare two therapeutics strategies: a promoted strategy and an alternative one. First results are
promising and reveal that the alternative strategy is more efficient in choosing the right drug. The
importance of such a result resides in the fact that it is possible to anticipate the public health impact
of the application of a medical strategy. On the other hand, this concerms the relevance of multi-agent
approaches for health care applications.
Keywords: Multi-agent systems, rule-based systems, integrative approaches, personalized medicine,
illustrating therapeutic strategies.
A.2 - Pairing Single Contact Nodes To Hub Regulates Protein Fold Plasticity
M. Achoch1, G. Feverati2, L.Vuillon2, K. Salamatian1, C. Lesieur2
1
LISTIC Université de Savoie, Annecy-le-Vieux, France ; 2FR 2914 (LAMA, LAPTH, AGIM) CNRS
Université de Savoie, Annecy-le-Vieux, France
Background: In recent years, protein structures have been investigated using graph theory applied on
networks of amino acids in interaction, the nodes being the amino acids and the links being the
interactions.
Objectives: We work on protein oligomers which associate several chains to carry out their biological
function. The association takes place through interactions between amino acids of different chains
making interface areas. Here we investigate the network features that provide the chain association its
robustness and its plasticity.
Methods: Inter-chain networks are constructed with our algorithm Gemini which reads the geometry
of the interfaces by measuring mutually nearest atoms between two chains. Our algorithm SpectralPro
based on spectral graph properties is used to analyze the networks.
Results and conclusion: The robustness means the resistance to random mutation and attacks against
specific nodes. The 1000 inter-chain networks studied have exponential degree distribution, avoid
hubs and favor many few link nodes instead. This leads to networks with low interconnectedness
which limits the spread of changes upon amino acid mutation because nodes are secluded from one
another (disconnected graph). The plasticity is the flexibility towards conformational changes needed
for the assembly of the protein. It is regulated by the pairing of a single contact node of a chain M with
a hub node on a chain M+1. The hub node yields further links to amino acids on the chain M, which
are far away on the protein sequence and bring them together in space, as such it regulates essential 3D
organization of the protein. Yet the vulnerability to mutation and the subsequent conformational
changes lies on the single contact node amino acid. The mutations of the amino acid arginine 337
(R337) or of the amino acid glycine 334 (G334) in the tumor suppressor p53 tetramer are extremely
common in some cancers. The mutations lead to a loss of the p53 function due to p53 fiber formation.
We report a network analysis of the p53 tetramer which shows that R337 and G334 are single contact
nodes paired with hubs. Using symmetry and 2D-tiling we propose a mechanism which explains how
the mutation leads to the conformational plasticity to go from the tetramer to the fiber.
A.3 - Bacterial profile and drug susceptibility pattern of neonatal sepsis in Gondar
University Hospital, northwest Ethiopia.
G Amare 1,3, L Wubishet 2, M Feleke 1, M Beyene 1, A Belay 1, Y Gizachew 1, N Tesfaye1, U
Chandrashekhar 1 and K Afework 1
1
School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences,
University of Gondar, P.O. Box 196, Gondar, Ethiopia.
2
Department of Pediatrics and Child Health, School of Medicine, College of Medicine and
Health Sciences, University of Gondar P.O. Box 196, Gondar, Ethiopia.
3.
Department of Viroscience, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands.
Background: Neonatal sepsis is a serious bacterial infection of blood with a clinical syndrome
characterized by systemic signs of infection and bacteremia in the first month of life.
Objective: We aimed to determine the bacterial profile drug susceptibility pattern of neonatal sepsis in
patients seen at Gondar University Hospital neonatal unit, Northwest Ethiopia.
Design: A cross sectional study was conducted involving patients with clinical signs of sepsis who
were admitted to Gondar University Hospital neonatal unit between July 2011 and June 2012. Venous
blood sample was collected aseptically and inoculated in to Trypton soya blood culture medium for 214 days aerobically, checking for growth of bacteria every 3 days. Bacterial isolates were identified
using standard procedures and antibacterial susceptibility test was done following agar disc diffusion
method.
Result: A total of 181 neonates (99 male and 82 female) were included in the study. Out of this, 58
showed positive blood culture for bacteria with infection rate of 32.1%. The most common bacterial
isolate was Staphylococcus aureus (29.3%) followed by Klebsiella ozaenae (17.2%), Escherichia coli
(10.3%), non-lactose fermenter gram negatives (10.3%), K. pneumoniae (8.6%) and coagulase
negative staphylococci (6.8%). S. aureus showed high level of drug resistance for tetracycline (16,
94.1%), ampicilln (14, 82.4%), amoxicillin (14, 82.4%), chloramphenicol (4, 82.4%), trimetoprim
sulphametoxazole (14, 82.4%) and ceftrioxone (13, 76.4%). Among the total isolates of S. aureus,
41.2% of them were found to be methicillin resistant (MRSA). K. ozaenae showed extremely high
level of resistance against amoxicillin (10, 100%), chloramphenicole (10, 100%), ampicillin (9, 90%)
and tetracycline (7, 70%). Multiple drug resistance was observed in 85.7% of gram positive and 92.1%
of gram negative isolates.
Conclusion: There is exceedingly high rate of resistance of bacterial isolates to different antibiotics
commonly prescribed in this region.
Acknowledgement: we thank all participants of this study.
Reference: Dramstadt GL, Black RE, Santosham M. Pediatr Infect Dis J 2000; 19: 739-750.
A.4 - Computational prediction of metabolic phenotypes from metabolomic data among
cancer cell lines
M.K. Aurich and I. Thiele
Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belval, Luxembourg.
The link between cancer and metabolism is well established. Mutations during transformation cause
the rewiring of the metabolic pathways, however differences in cancer metabolism have been
observed.
A genome-scale metabolic reconstruction provides a framework to investigate mechanisms underlying
the genotype-environment-phenotype relationship. Analysis of complex omics data sets in the network
context, especially metabolomics data offer great potential for the clinic, since metabolomic profiles
can easily be derived from body fluids of patients. However, methods for the integration of
metabolomics data sets with the human metabolic reconstruction are less well developed.
Herein, we used published metabolomics data and a novel computational method to analyze
metabolomics data in the network context to generate condition-specific metabolic models for the
NCI-60 cell lines. The metabolic models were generated such that they are consistent with the
measured data and known human biochemistry. Consequently, the majority of models were able to
realize experimental growth rates. We used this compendium of metabolic models to explore the
metabolic strategies followed by the different cell types. For instance, we observed a large span of
ATP yields per unit of glucose (0-42) among the cell line models, pointing toward the existence of
different routes to supply the cells with energy. We explored conditionally essential genes in these cell
line models to further understand how different their use of metabolism is. In only a few of the
models, genes of the TCA cycle were essential. For example, the in silico knock-out of isocitrate
dehydrogenase disabled growth in four models. Further, we identified phosphoglycerate
dehydrogenase was essential for eleven of the generated models. These predictions do not only
illustrate differences in metabolic strategies but suggest also novel drug targets specific to a cancer
type. The models also differed on their requirements for oxygen uptake rate, and required either high
or low rates, or where indifferent about the rates of oxygen supplied. Comparison of the contribution
of ATP, NADH, NADPH and FADH2 producing reactions among the models further supported
different metabolic phenotypes.
Taken together, our predictions characterize the metabolic heterogeneity among the NCI-60 cell lines,
and provide potential drug targets for the distinct phenotypes. Since our methods allow a relatively fast
overview of cellular metabolic traits, they could be readily applied to investigate other diseases.
A.5 -
Clinical Utility of a Coronary Heart Disease Risk Gene Score in a Pakistani Population
KE Beaney1, W Ahmed2, P Howard1 & SE Humphries1.
1
Centre for Cardiovascular Genetics, BHF Laboratories, University College London, London, UK
Department of Microbiology, Haripur University, Haripur, Pakistan.
2
Background: Numerous risk prediction algorithms based on conventional risk factors for Coronary
Heart Disease (CHD) have been developed. However, these only provide modest discrimination (1)
and novel risk factors for inclusion are being investigated. One candidate for this is genetics.
Therefore, a CHD-risk gene score, which determines genetic CHD risk based on 19 SNPs has been
developed. It was assessed using data from the Northwick Park Heart Study II (NPHSII) - a
prospective CHD study of approximately 3000 Caucasian men- and found to have clinical utility
(Drenos et al, unpublished). However, given that linkage disequilibrium patterns differ between ethnic
groups it is unknown whether the gene score would have clinical utility in other ethnic groups.
Objective: The objective was to investigate whether a CHD-risk gene score had clinical utility in a
Pakistani sample set.
Design: In order to investigate our objective, the nineteen SNPs from the gene score were genotyped
in a Pakistani sample set (n=549, 321 cases and 228 controls) using KASPar and Taqman genotyping
assays.
Results: Genotyping has been completed for fifteen SNPs. Of these, the genotypes for five SNPs
were out of Hardy-Weinberg equilibrium. In each case this was due to an excess of rare homozygotes.
The most likely explanation for this is consanguinity in this population. The frequencies of the risk
alleles for each SNP were then compared for the Pakistani sample set control samples and NPHSII.
There was a significant difference (p-value <0.05) between the risk allele frequencies for ten of the
SNPs. In some cases, the risk allele was more frequent in the Pakistani sample set and in some cases it
was less frequent.
Conclusions: Significantly different allele frequencies were found between a Pakistani sample set and
Caucasian sample set for SNPs in the CHD risk gene score. Once the remaining genotyping has been
completed, the mean gene score will be determined for both cases and controls, with the hypothesis
that it should be significantly higher for the cases.
Acknowledgements: Thanks are offered to Waqas Ahmed who provided the samples in the Pakistani
sample set.
KB is funded by an MRC CASE award in conjunction with RANDOX.
References: 1. Cooper JA, Miller GJ, Humphries SE. A comparison of the PROCAM and
Framingham point-scoring systems for estimation of individual risk of coronary heart disease in the
Second Northwick Park Heart Study. Atherosclerosis. 2005;181(1):93-100.
A.6 - Atherogenic biomarkers in vascular dementia: the role of inflammation in
neurodegeneration
JC Carril and R Cacabelos
Chair of Genomic Medicine, Camilo José Cela University, Madrid, Spain
Background: The molecular basis for the genetic risk of cerebrovascular disease is likely to be
multigenic and influenced by environmental factors. The influence of cytokine gene polymorphisms in
modulating the inflammatory pattern involved in forming clots that could trigger arterial ischemic
processes has been postulated.
The ability to identify high-risk patients through genetic testing could make screening for treatable
intermediate phenotypes more cost-effective.
Objective: The aim of this study was to evaluate the role of specific SNPs in the interleukin genes
IL1B (3954T/C), IL6 (-174G/C and -573G/C), IL6R (1510A/C) and TNFA (-308G/A) in vascular
dementia and other cerebrovascular-related pathologies.
Design: A total of 5 genetic variants in 4 different inflammation-related genes were determined in 457
individuals, 153 patients with vascular dementia (VaD), 148 with vascular encephalopathy (VaE), 46
stroke patients (CVA) and 110 control subjects without ischemic or hemorrhagic stroke history, or
other cerebral diseases, aged over 50 years.
Genotyping was performed using TaqMan® OpenArray® DNA microchips (Life Technologies).
DNA was extracted from whole blood using Qiagen extraction columns.
The standard chi-square method was used to test deviation from the Hardy-Weinberg equilibrium
(HWE) and to compare genotypic and allelic frequencies in vascular and control groups. Additionally,
odds ratios were calculated, and their appropriate confidence intervals were computed at the 95%
level.
Results: The most informative genotypes in vascular dementia were IL6*-573GG (29.70% vs.
68.29%, p<0.001) and IL6R*1510AA (25.74% vs. 45.12%, p<0.001). Relevant genotypes for VaE
were IL6*-573GG (29.70% vs. 51.67%, p<0.001) and IL6R*1510AA (25.74% vs. 45.00%, p<0.001).
Conclusions: The results found in this preliminary study show the relationship between the plasmatic
levels of cytokines and the major incidence of cerebrovascular accidents. Increased levels of IL6 and
its receptor IL6R increase the recruitment of monocytes and macrophages to the lesion-prone sites,
contributing to the formation of the atheroma plaque.
We confirmed a strong association with IL6 and IL6R variants in our patients. Polymorphisms for this
panel of cytokine genes are a valuable tool to assess the risk of vascular dementia and to help us
identify high-risk profiles.
Acknowledgements: This investigation was partially supported by a grant from the Camilo José Cela
University (Genescreen project 2014-2015).
A.7 - Molecular Predictors of Antidepressant Treatment Outcome
T Carrillo-Roa1, CA Lareau2, CL McGrath3, BW Dunlop3, ME Kelley3, EB Binder1,3, HS Mayberg3,4.
1. Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Munich,
Germany. 2. Department of Mathematics, University of Tulsa, Tulsa, United States of America. 3.
Department of Psychiatry and Behavioural Sciences, Emory University, Atlanta, United States of
America. 4. Department of Neurology, Emory University, Atlanta, United States of America.
Background: Major depression is a prevalent disease with high rates of treatment resistance and nonremission. Recently, our group described that resting state brain activity patters of six specific brain
regions (ROIs) can predict differential response to either escitalopram (sCIT) or cognitive behavioural
therapy (CBT) (1).
Objective: The aim of this study is to identify molecular markers that associate with these brain
activity patterns, in the hope to identify predictive measures that are more easily obtained in clinical
practice than neuroimaging measures. Design: Patients were recruited at Emory University and
randomized at baseline to 12 weeks sCIT, or 16 sessions of CBT. Genome-wide genotypes (Illumina
OmniExpress) and DNA methylation (Illumina HM-450K) were measured in peripheral blood DNA
drawn at baseline. Genome-wide SNPs and CpGs univariate and multivariate association analyses
including ROIs combinations were conducted in 76 MDD patients.
Results: We observed genome-wide significant association of rs34383296 (p = 9.4x10-9) in a
multivariate analysis that included three brain regions. Univariate analyses did not reveal genomewide significant associations. The associated variant lies in a gene dense region on chromosome 9
within the NDOR1 gene and it is an eQTL for ARRDC1, a gene ~400kb downstream related to
arrestin-mediated internalization of cell surface receptors. This SNP was genotyped in an independent
larger MDD sample and predicted differential response to CBT vs. drug (sCIT and Duloxetine). No
epigenome-wide significant association was observed.
Conclusions: Our data suggest that using quantitative neuroimaging endophenotypes and genomic
approaches may be able to identify markers to guide individualized depression therapy choices.
1. McGrath CL, Kelley ME, Holtzheimer PE, Dunlop BW, Craighead WE, Franco AR, et al. Toward a
neuroimaging treatment selection biomarker for major depressive disorder. JAMA psychiatry
(Chicago, Ill). 2013;70(8):821-9.
A.8 - Vascular Risk Factors in Dementia: Influence of APOE, MTHFR, NOS3, PSEN1
and ACE Polymorphisms and Association with Cognitive Function Scores
L. Corzo, J.C. Carril, S. Rodríguez, V. Couceiro, R. Cacabelos
EuroEspes Biomedical Research Center, Santa Marta de Babío s/n. 15166-Bergondo, Corunna,
Spain
Background: Vascular dysfunction is associated with the pathogenesis of dementia; however, some
studies have shown unclear evidence for an association between vascular risk factors and poorer
cognitive performance.
Design: In order to evaluate the possible association between cognitive status and emergent vascular
risk markers (EVRM), we analyzed ultrasensitive C-reactive protein (us-CRP), fibrinogen (F),
lipoprotein (a) [Lp(a)] and homocysteine (HCY) concentrations in serum/plasma from 30 patients with
dementia and 62 non-demented control subjects. MMSE, ADAS-cog and GDS scales were used to
evaluate cognitive function. A genetic vascular risk panel was included to observe its influence on
EVRM. APOE, MTHFR C677T, NOS3 G894T, PSEN1 Pol and ACE Ins/Del genotyping was
performed.
Results: Folate levels in healthy subjects were negatively associated with HCY, and us-CRP
positively correlated with F in the two groups. These correlations were independent of APOE and
MTHFR polymorphisms. Data did not show an association between EVRM and cognitive
performance. To find changes in EVRM between mild cognitive impairment (MCI) and dementia, we
classified patients as controls, MCI and dementia. Higher concentrations of HCY were found in
dementia versus controls (p< 0.02) and MCI (p< 0.03). Lower us-CRP levels were observed in
dementia versus control (p< 0.02) and MCI (p< 0.02). No significant differences were observed in the
other EVRM. Lp(a) levels were higher in APOE4 carriers (p= 0.031). Regarding the MTHFR C677T
polymorphism, CC carriers showed lower levels of us-CRP than CT carriers.
Conclusions: Based on these findings, we conclude that:
i) Inflammation, atherothrombotic and HCY-related vascular mechanisms seem to be associated with
dementia, and these mechanisms are interrelated with each other;
ii) Only us-CRP and Lp(a) concentrations in serum are affected by the genetic risk markers analyzed;
and
iii) No association exists between EVRM and cognitive function scores.
Reference: Cacabelos R, Fernández-Novoa L, Lombardi V, Corzo L, Pichel V, Kubota Y.
Cerebrovascular risk factors in Alzheimer's disease: brain hemodynamics and pharmacogenomic
implications. Neurol Res 2003 Sep;25(6):567-80.
A.9 - Relationships between Genetic Vascular Risk Polymorphisms and Serum Vascular
Biomarkers
L. Corzo, S. Rodríguez, J.C. Carril, R. Cacabelos
EuroEspes Biomedical Research Center, Santa Marta de Babío s/n. 15165-Bergondo, Corunna, Spain.
Background: The progressive increase in vascular diseases in developed countries and the knowledge
that vascular risk is involved in numerous pathologies have generated a great interest in the
development of predictive vascular markers. Tracing both the human genome and protein expression
to ascertain the probability of risk of vascular pathologies can be definitive in controlling the disease.
Objective: Our goal was to find the effect of genetic vascular risk polymorphisms on the serum
vascular biomarkers used in clinical practice.
Design: The study was conducted in 199 persons [111 (55.8%) men; 88 (44.2%) women; age:
59.8814.41 years] who presented any vascular symptom or sign. Different mechanisms involved in
vascular dysfunction, such as dyslipidemia, endothelial function, hypertension, immune response and
thrombosis, were assessed by the following serum parameters: total cholesterol (TC), HDL-cholesterol
(HDL), LDL-cholesterol (LDL), triglycerides (TG), apoA1, apoB, apoE, folate (F), vitamin B12 (B12),
homocysteine (HCY), fibrinogen (FB), lipoprotein (a) (LP), ultrasensitive-PCR (PCR) and atherogenic
indices. Genetic vascular-associated polymorphisms included: APOC3 C3175G, APOB C7673T,
APOE C112T/C158T, CETP G+279A, LPL C1421G, NOS3 G894T, ACE Alu 287bp indel (I/D), AGT
M235T, IL1B T3954C, IL6 G-174C, IL6R A1510C, TNFA G-308A, F2 G20210A, F5 G1691A, and
MTHFR C677T.
Results: We found higher LDL levels in patients carrying APOE allele4. Higher TG concentrations
and apoB/apoA1 index were observed in patients with the APOB T7673T genotype. Subjects
presenting CETP GG had lower levels of HDL and apoA1. ACE D/D genotype was associated with
lower levels of TC and TC/HDL index. AGT T235T genotype was associated with decreased FB and
PCR levels. IL6R A1510C polymorphism was correlated with higher concentrations of B12. F5
G1691A polymorphism was found in patients with higher levels of HDL, apoA1, TC and LP.
Conclusions: Lipid dysfunction-related genotypes (APOE allele4, APOB T7673T, CETP GG) were
associated with increased atherogenic and decreased atheroprotective lipid levels. However, ACE D/D,
AGT T235T and IL6R A1510C genotypes, involved in endothelial impairment, hypertension and
immune response respectively, were not associated with an atherogenic effect. Coagulation-involved
F5 G1691A genotype, only present in 3 subjects, was associated with both atheroprotective and
atherogenic factors.
The results could support that:
i) Pure atherogenic mechanism is only related to lipid metabolism-dependent genotypes.
ii) Genome and lipidome/proteome might generate an independent but additive risk for developing
vascular disease.
Reference: Vasan RS. Biomarkers of cardiovascular disease: molecular basis and practical
considerations. Circulation 2006 May 16;113(19):2335-62.
A.10 - Effects of 17-beta estradiol on proliferation and cardiac integration of human
Wharton’s jelly-derived mesenchymal stem cells
M.C. Corotchi1*, M.A. Popa1*, M. Simionescu1
1
Angiogenesis and Cardiovascular Remodelling Group, Department of Regenerative Medicine,
Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy
*These authors contributed equally to this work
Introduction: Among multiple sources of stem cells, mesenchymal stem cells (MSCs) obtained
from human umbilical cord matrix, namely Wharton’s jelly (WJ), are promising candidates in
the emerging field of regenerative medicine. MSCs have been employed in various
experimental cardiac applications providing an interesting therapeutic potential. Since estrogens
are reported to be beneficial for a range of cardiac diseases, the objective of our study was to
investigate the effects of 17-beta estradiol (E2) on adult MSCs proliferation and tissue
integration and define the cellular/molecular mechanisms involved in cardiac
integration/regeneration.
Materials and Methods: WJ was isolated from the umbilical cords after dissection and removal of
the umbilical cord arteries, vein and amniotic epithelium. MSCs isolation from WJ was achieved
either by mechanical dissociation or by enzymatic dissociation. An impedance measurement-based
Real-Time Cell Analyzer (xCELLigence system, Roche) was used to investigate the effect of different
E2 concentrations on WJ-MSCs proliferation and chemotaxis. The WJ-MSCs migration/integration
towards/into the cardiac tissue was studied employing adult-heart murine ventricular slices cocultured
with WJ-MSCs. Quantitative determination of the gene expression of proliferation/angiogenic markers
was done on MSCs stimulated or non-stimulated with E2 employing RT-PCR and qRT-PCR.
Results: Exposure of WJ-MSCs to E2 up to 100nM concentration had no cytotoxic effect on cells.
Furthermore, as compared to non-stimulated cells, exposure to E2 of WJ-MSCs induced a higher
proliferation rate (~ 50%) than that detected in non-stimulated cells after six days in culture. In
addition, the chemotaxis assays revealed an increased cell migration and integration towards/into the
murine adult-viable heart ventricular slices in comparison with the non-stimulated cells. A dose/timedependent modulation in gene expression of MMP-2, Angiogenin, Cav-1, Cav-2, EMMPRIN,
Endostatin was detected in E2-exposed MSCs.
Conclusion: The effects of 17-beta estradiol on WJ-MSCs are: increased proliferation rate and life
span of the cells; higher capacity of integration and migration towards/into the viable heart ventricular
slices and the modulation of the gene expression of proliferation/angiogenic markers. Understanding
the fine mechanisms underlying these effects would be of great help for optimizing cell therapy
approaches for regenerative medicine.
Acknowledgements: This work was supported by Swiss-Romanian Cooperation Programme
SNSF Grant No. IZERO_142213/1 and CNCS-UEFISCDI/RSRP No 21 and by the Romanian
Academy.
Keywords: Wharton’s jelly, mesenchymal stem cells, 17-beta estradiol, coculture, tissue slices.
A.11 - Evaluation of formaldehyde exposure in the work environment - biomarkers of effect
and susceptibility.
S Costa1, LS Santos2,3, C Costa1, S Silva1, J Gaspar2, B Porto4, B Laffon5, JP Teixeira1.
1
National Institute of Health, Porto, PORTUGAL; 2 New University of Lisbon, Lisboa, PORTUGAL;
Portuguese Catholic University, Viseu, PORTUGAL; Institute of Biomedical Sciences Abel Salazar,
Porto, PORTUGAL; 5University of A Coruña, A Coruña, SPAIN.
3
Background: Formaldehyde (FA) is a high-volume production chemical produced worldwide with a
large range of industrial and medical uses. Over the last two decades, several epidemiological studies
have revealed an increased risk of cancer development among FA-exposed workers, namely
nasopharyngeal cancer and myeloid leukaemia. Based on these findings plus supporting evidence from
animal studies and data on mechanisms of carcinogenesis, FA status was recently revised and
classified as a human carcinogen.
Objective: The aim of the present study was to evaluate the occupational exposure to FA in a
multistage approach relating exposure with effect biomarkers and individual susceptibility.
Design: Eighty-five FA-exposed workers from hospital anatomical pathology laboratories and eightyseven non-exposed controls took part in the study. Air monitoring was performed in worker’s
breathing zone for representative working periods and the level of FA-exposure in workplace air was
estimated. Cytogenetic damage was assessed by means of micronucleus and DNA damage by comet
assay (percentage of tail DNA). In addition, the effect of polymorphic genes of xenobiotic
metabolising enzymes (CYP2E1, GSTM1, GSTT1, GSTP1) and DNA repair enzymes (FANCA,
RAD51, XRCC2, XRCC3, XRCC1, PARP1, MUTYH, BRIP1) on the endpoints studied was
determined.
Results: The mean level of FA-exposure was 0.38±0.03 ppm, exceeding recommended limit values, as
observed in other studies. All genotoxic endpoints were significantly increased in FA-exposed workers
compared to controls. Concerning the effect of susceptibility biomarkers on the different endpoints
studied, results suggest that polymorphisms in metabolic genes are associated with increased genetic
damage in FA-exposed subjects. Furthermore, a polymorphic gene involved in FANC repair pathway,
FANCA, significantly altered the level of genetic damage induced by FA exposure, revealing a
potential novel repair pathway involved in the repair of genetic lesions caused by FA-occupational
exposure.
Conclusions: Overall, this work highlights the contribution of genetic variation on metabolic and
DNA repair genes to the interindividual variability in susceptibility to FA-induced genetic damage,
reinforcing the need to further implement toxicogenomic association studies.
Acknowledgements: This work is supported by Fundação para a Ciência e Tecnologia under the
grants SFRH/BD/46929/2008 and PTDC/SAU-ESA/102367/2008.
A.12 - Progression of abdominal obesity in youth followed by testosterone decrease leading to
erectile dysfunction and sterility
V Dimitrijevic-Sreckovic1,4 , H Janeski1, B Sreckovic2, I Soldatovic3,4, J Gacic2, D Mitrovic1, P
Djordjevic1,4
1
Clinic for Endocrinology, Diabetes and Metabolic Diseases,2 Bezanijska kosa Clinical Center,
Institute of Medical statistics and Informatics,4Faculty of Medicine, University of Belgrade, Serbia
3
Background: Visceral obesity, which in fact is a metabolic syndrome (MS), is related to decreased
testosterone, insulin resistance (IR), increased inflammatory factors, fat deposition in the liver and
occurrence of non-alcoholic fat liver disease (NAFLD). Elevated inflammatory markers (CRP) may
interfere with insulin signal transduction at the neuronal level inducing hypogonadotropic
hypogonadism.
Objective: To examine testosterone relationship with abdominal obesity, lipid status, blood pressure,
IR and NAFLD in obese young males with pre-MS and MS.
Design: Cross-sectional study.
Methods: The study included 52 obese male individuals with pre-MS or MS (age16-30) classified into
two groups: I-with low testosterone <12.0nmol/l; II-with testosterone ≥12,0nmol/l. The following
parameters were observed: waist circumference (WC), blood pressure, lipids, microalbuminuria.
SGOT, SGPT and γ-GT were liver function parameters. ATP III classification was applied for
diagnosing MS. Patients with less than three above criteria were considered pre-MS. IR was
determined by HOMA IR. OGTT (0, 30 and120 minute glycaemia and insulin) was used to evaluate
the
extent
of
disorder.
Testosterone
was
determined
by
radioimmunoassay.
Results: Results for groups I and II were as follows:BMI:I-35.7±35, II-33.0±4.9kg/m2;WC:I117.3±15.5, II-109.9±14.2 cm; HDL:I-0.96±0.18,II-1.04±0.2mmol/l; triglycerides:I-2.74±1.6,II1.8±1.0mmol/l; insulin 0 min:I-113±128.II-40.1±61.2IU/l; insulin 30 min:I-199.8±124.II124.1±90IU/l;
insulin
120
min:I-119.8±114.II-53.9±70.8IU/l;
HOMA
IR:I-26.8±31,II
9.1±14µmol/mU/ml; SGOT:I-50.5±39.3,II-26.8±7.8;SGPT:I-81.8±48.2, II-40.2±18.0 U/l; γ-GT:I49.8±19.3U/l, II-39.7±21.9 U/l. CRP :I-5.2±2.5, II- 5.25±5.8 mg/l. Correlations : testosterone negative
with body weight, BMI and WC (p<0.05). Decreased testosterone (<12.0nmol/l) was found in 13.5%
obese young males (8.5±2.6nmol/l), with normal FSH, LH and estradiol. A statistically important
difference between groups was found for 0, 30 and 120 minute insulin values (p<0.05) and for liver
function parameters SGOT and SGPT (p<0.001).
Conclusion: Low testosterone is characterized by obesity, MS parameters, hyperinsulinism, IR and
NAFLD. Negative correlation of testosterone with WC and statistical importance of insulinemia and
liver function parameters differences confirm the important effect of visceral obesity and IR on the
occurrence of erectile dysfunction and NAFLD in obese adolescents and youth.
Reference: Wang C., Jackson G., Jones T.H., et al. Low Testosterone Associated With Obesity and
the Metabolic Syndrome Contributes to Sexual Dysfunction and Cardiovascular Disease Risk in Men
With Type 2 Diabetes.Diabetes Care 2011;34:1669-1675
A.13 - Decreased insulin sensitivity and antioxidant status and increased thrombotic and
inflammatory factors in obese children, adolescents and youth
V Dimitrijevic-Sreckovic1,5, B Sreckovic2, H Janeski1, E Colak3, I Soldatovic4,5, P Djordjevic1,5
1
Clinic for Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Serbia 2Bezanijska
Kosa Clinical Center, 3Institute of Medical Biochemistry, 4Institute for Medical Statistics and
Informatics, 5Faculty of Medicine, Belgrade University, Serbia.
Background: In obesity and metabolic syndrome (MS) abdominal obesity is accompanied with
hyperinsulinism and insulin resistance (IR). As IR is increasing, insulin sensitivity (IS) is decreasing.
We used Matsuda index of whole-body insulin sensitivity derived from the oral glucose tolerance test
(OGTT), which represents a composite of both hepatic and peripheral tissue sensitivity to insulin.
Abdominal obesity also correlates with increased thrombotic and inflammatory factors supporting the
progression of atherosclerotic complications.
Objective: This study analyzes IR and IS, thrombotic and inflammatory factors, and antioxidant status
in obese children, adolescents and youth.
Methods: The study included 205 obese individuals (age 7-30) classified according to age I-children
(7-15), II-adolescents (16-20) and III-youth (20-30). IR was determined by homeostatic model
assessment (HOMA IR). IS was determined by Matsuda index of whole-body insulin sensitivity (10
000/square root of (fasting glucose x fasting insulin)x(mean glucose x mean insulin during OGTT).
Activities of markers of antioxidant defense, superoxide dismutase (SOD) and glutathione peroxidase
(GPX) were determined in erythrocytes.
Results: MS increases considerably with age, was found in 33.3% children, 46.2% adolescents and
50.5% youth. Patients had increased waist circumference (WC):(I-90.3±10.9, II-104.2±15.4, III105.2±20.2 cm), blood pressure (I-113.0±13.2 /75±9.4, II-121.9±12.4/81.9±9.93, III126±19.0/82.7±10.7mmHg), HOMA IR (I-5.6±2.8, II-7.5±12.9, 9.2±13.5), triglycerides (I-1.4±0.6, II1.5±0.7, III-1.9±1.8mmol/l), CRP(I-2.8±6.2, II- 6.5±8.2, III-7.4±4.4mg/l), PAI-1 (I-5.7±2.2 II6.3±1.2,III- 6.4±1.1 U/ml) and microalbuminuria (I-29.4±20.3, II-43.3±31.1,III- 40±30.1mg/24h), and
decreased HDL (I-1.17±0.2, II-1.15±0.2, III-1.14±0.4mmol/l), SOD (I-1057.5±147.2, II1125.3±215.5,III- 1115.5±178.9 U/gHb), GPX (I-29.6±3.2, II-19.7±15.4, III-33.2±11.1U/gHb), and
decreased Matsuda index (I-4.7±3.2, II 5.6±4.6, III-5.4±4.5). Correlations:HOMA IR with BMI, WC,
blood pressure, triglycerides, basal, 30 min, 120 min and mean insulinemia, basal glycemia (p<0.01),
PAI-1 (p<0.05), CRP (p<0.01) and negatively with HDL (p<0.01). Patients with MS had increased IR
(HOMA IR 12.1±16.6) and decreased IS (Matsuda index 3.7±3.2) in regard to patients without MS.
Conclusion: In obese patients, abdominal obesity is accompanied with hyperinsulinism, increased IR,
decreased IS and antioxidant status. Correlations of HOMA IR with WC, PAI-1 and CRP explain
connection between IR and abdominal obesity, increased levels of thrombotic and inflammatory
factors and early risks of atherosclerosis.
A.14 - Prebiotic properties of oligo(2-7)-galacturonic acid on mouse gut microbiota1
M A Engevik §,*, C J Faletti+, R T Worrell §,* and M Paulmichl+
§Department of Molecular and Cellular Physiology University of Cincinnati College of Medicine
Cincinnati, OH 45267
*Digestive Health Center of Cincinnati Children’s Hospital, Cincinnati, OH 45229
+Institute of Pharmacology & Toxicology, Helga & Erich Kellerhals Laboratories for Novel
Therapeutics, Center for Pharmacogenetics and Pharmacogenomics, Paracelsus Medical University
Strubergasse 21, 5020 Salzburg, Austria
Background and Objectives: With the rise of antibiotic resistance, new alternatives are being sought
to effectively modulate the characteristics of gut microbiota to obtain pathogen resistance without the
use of antibiotics. In the past, an oligosaccharide derivative of carrots, oligo(2-7)-galacturonic acid
(GHF7K), has been used clinically and recently in the fowl-industry to promote health. This study
examined the potential role of GHF7K as a prebiotic to alter the gut microbiota in mice. Design: Mice
were fed either a control diet or a diet containing 2% GHF7K in the water and chow for 2 weeks, and
weight, food and water consumption, gut microbiota and ion composition of the intestinal fluid were
examined. Results: Dietary supplement with GHF7K did not alter mouse weight or daily food
consumption. Additionally, no changes were observed in the total number of luminal or mucosaassociated bacteria populations in GHF7K-fed mice. However, GHF7K supplementation significantly
altered the composition of luminal, and to a less extent, mucosa-associated bacterial populations at the
level of the phyla, with region-specific differences. Similar to antibiotic use, Proteobacteria number
was increased in the ileum and colon of GHF7K–fed mice, with no changes in the number of
beneficial Lactobacillus and Bifidobacterium genera of phylum Firmicutes. Corresponding with the
altered gut microbiota, changes in the ion composition of the intestinal fluid were observed. An
increased Cl- concentration was observed in the duodenum and jejunum, while the Na+ concentration
was increased in the cecum of GHF7K-fed mice. Decreases were observed in the K+ concentration in
the cecum and distal colon.
Conclusions: Dietary supplement of GHF7K is capable of altering the gut microbiota, which
correlates to changes in the intestinal environment. These data suggest that GHF7K dietary
supplement can purposefully be used to alter the gut microbiota, and thus could represent an
alternative approach to prophylactic antibiotic use.
References: 1. Engevik, M.A., Faletti, C.J., Paulmichl, M. & Worrell, R.T. Prebiotic Properties of
Galursan HF 7K on Mouse Gut Microbiota. Cell Physiol Biochem 32, 96-110 (2013).
A.15 - DNA diagnostics of MIDD and MELAS syndromes in Slovakia
D. Gasperikova1, M. Skopkova1, I. Masindova1, L. Valentinova1, J. Stanik1,2, L. Varga
Huckova1, D. Danis1, Slovak MIDD/MELAS study group, M. Profant3, I. Klimes1
1,3
, M.
1
DIABGENE & Diabetes Laboratory, Institute of Experimental Endocrinology, SAS, Bratislava,
Slovakia,
2
First Department of Pediatrics, School of Medicine, Comenius University, Bratislava, Slovakia;
3
First Department of Otorhinolaryngology, School of Medicine, Comenius University, Bratislava,
Slovakia
The two syndromes, MIDD (Maternally Inherited Diabetes and Deafness) and MELAS (Mitochondrial
Encephalopathy, Lactic Acidosis and Stroke-like episodes) arise on a common genetic cause –
a mutation in mtDNA, most often m.3243A>G in the gene for tRNALeu. This mutation leads to
different clinical symptoms according to heteroplasmy levels in different tissues. Usually, the first
presentation of the MIDD syndrome is a progressive bilateral sensorineural hearing loss emerging in
adolescence. Diabetes develops mostly in 30 – 40 years of life with clinical picture resembling type 2
diabetes. The MELAS syndrome has a more severe progression with further neurological and
metabolic symptoms.
We tested 317 patients from 257 families with suspected MIDD or MELAS syndromes fulfilling
criteria of matrilineal inheritance, conjoint diabetes and hearing impairment, diabetes development
after 25th year of life, or progressive hearing loss. Patients´ DNA was extracted from peripheral blood
and/or buccal mucosa and analysed for presence of m.3243A>G variant using RFLP and/or Real-Time
PCR.
The m.3243A>G mutation was found in 18 patients from 8 families. The heteroplasmy was higher in
DNA samples from buccal swabs compared to blood DNA samples. In one case, the heteroplasmy was
detected in the buccal DNA only, while the DNA sample gave negative results repeatedly. Patients´
phenotypes varied from diabetes as the sole symptom to a complex picture of the MELAS syndrome.
DNA testing helped to determine the diagnosis, thus permiting correct patient management and
intensified surveillance of yet healthy mutation cariers.
Supported by APVV 0148-10, „KCMM“ (ITMS 26240220071)
A.16 - Gene polymorphisms predisposing to type II diabetes mellitus and/or obesity in 513
Greeks: Frequency distribution of TCF7L2, MTNR1B, CDKAL1, SLC30A8 and FTO
S.F. Gourzi, K. Tentolouris, N. Drakoulis
National and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy
Background and Objectives: Diabetes mellitus type 2 is a chronic metabolic disease with pandemic
spread worldwide. Environmental influences and genetic background are included in the pathogenesis
of the disease. More than 40 suspected loci have been associated with the risk of developing type 2
diabetes. Four of these (rs7903146, rs10830963, rs7756992, rs13266634) were studied in a sample of
Greek population. Obesity is also a chronic disease and is the most important risk factor for type 2
diabetes. Many SNPs have been associated with obesity with predominant the FTO rs9939609, which
has been studied in the same sample.
Methods: For the present study were studied 513 volunteers. The DNA used was collected from
buccal epithelial cells of the oral cavity of the volunteer. It was isolated by the extraction method and
measured by the method of Real Time PCR.
Results: The results have shown that the frequency of alleged dangerous alleles is ranging from 25%
to 70%. There were no differences in the frequencies between both sexes in genotypes and allele level.
The rs9939609 is quite frequent (44% frequency of A), without any differences in both sexes and has
also been associated with type 2 diabetes, although it is still unknown whether the association is
independent or not by obesity.
Conclusions: Although the genetic basis of type 2 diabetes and obesity is now proven, it is not yet
unfortunately been transferred in daily clinical practice. It is, however, expected in the following years
to contribute significantly to the prevention, diagnosis and treatment of these frequent diseases.
References: Gourzi S.F., Tentolouris K., Drakoulis N. Frequency distribution of polymorphisms of
TCF7L2, MTNR1B, CDKAL1, SLC30A8 and FTO genes predisposing to type II diabetes mellitus
and/or obesity in Greek population. In: N. Drakoulis (ed.) Contribution of Genetic Analyses in
Predisposition, Diagnosis, Prognosis and Therapeutic Approach to Disease, Chapter 3, G. Chrousos,
C. Liapi (eds.) Metabolic syndrome, nutrition and dietetics, asthma. APR-Publications, Athens 2013,
pp 92-100.
A.17 - Exhaustively characterizing feasible logic models of a signaling network using Answer
Set Programming
C. Guziolowski 1, S. Videla 2,3,4, F. Eduati 5, S. Thiele 3,2, T. Cokelaer 5, A. Siegel 2,3, and J. SaezRodriguez 5
1 Ecole Centrale de Nantes, IRCCyN UMR CNRS 6597, 1 rue de la Noe¨ , 44321, Nantes, France. 2
CNRS, UMR 6074 IRISA, Campus de Beaulieu, 35042 Rennes, France. 3 INRIA, Dyliss project,
Campus de Beaulieu, 35042 Rennes, France. 4 Universität Potsdam, Institut f ür Informatik, D-14482
Potsdam, Germany. 5 European Bioinformatics Institute, Wellcome Trust Genome Campus,
Cambridge CB10 1SD, UK.
Background: Logic modeling is a useful tool to study signal transduction across multiple pathways.
Logic models can be generated by training a network containing the prior knowledge to
phosphoproteomics data. The training can be performed using stochastic optimization procedures, but
these are unable to guarantee a global optima nor to report the complete family of feasible models.
This, however, is essential to provide precise insight in the mechanisms underlaying signal
transduction and generate reliable predictions.
Results: We propose the use of Answer Set Programming (ASP) to explore exhaustively the space of
feasible logic models. Towards this end, we have developed caspo, an open-source Python package
which provides a powerful platform to learn and characterize logic models by leveraging the rich
modeling language and solving technologies of ASP. We illustrate the usefulness of caspo by
revisiting a model of pro-growth and inflammatory pathways in liver cells. We show that, if
experimental error is taken into account, there are thousands (11,700) of models compatible with the
data. Despite the large number, we can extract structural features from the models, such as links that
are always (or never) present or modules that appear in a mutual exclusive fashion. To further
characterize this family of models, we investigate the input-output behavior of the models. We find 91
behaviors across the 11,700 models and we suggest new experiments to discriminate among them.
Conclusion: Our results underscore the importance of characterizing in a global and exhaustive
manner the family of feasible models, with important implications for experimental design.
Availability: caspo is freely available for download (license GPLv3) and as a web service at
http://caspo.genouest.org/.
Acknowledgements: We thank the financial aid from the EU through project “BioPreDyn” (ECFP7KBBE-2011-5 Grant number 289434), the French National Agency for Research (ANR-10-BLANC0218), and the Federal Ministry of Education and Research (BMBF “MEDSYS” project 0315401B).
Reference: Guziolowski C, Videla S, Eduati F, Thiele S, Cokelaer T, Siegel A, Saez-Rodriguez J.
Exhaustively characterizing feasible logic models of a signaling network using Answer Set
Programming. Bioinformatics 2013; 29(18) 2320-2326
A.18 - Histone acetylation regulates endothelial differentiation of fetal stem cells
1
F Iordache, 1E Andrei , 1A Constantinescu., 2 C Curutiu, 1H Maniu
Department of Regenerative Medicine, Institute of Cellular Biology and Pathology “Nicolae
Simionescu” of Romanian Academy, 050568, Romania
2
Department of Microbiology-Immunology, Faculty of Biology, University of Bucharest, 76201,
Romania
1
Introduction: Epigenetic changes in the genome include DNA methylation, histone modifications
(acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation), and
recently discovered miRNAs, three mechanisms that are often tightly linked in the regulation of gene
expression and involved in many cellular processes. Histone acetylation was seen as a phenomenon
correlated with an open chromatin conformation that allowed the expression of different genes
involved in differentiation. Currently it has been observed that in acetylated state, many genes are
repressed and thus differentiation to a specific cell line is blocked, maintaining the pluripotent state.
Our aim was to investigate the role of histone acetylation in differentiation of endothelial progenitor
cells.
Materials and methods: Caracterization of EPCs was performed by flow cytometry and
neovascularization potential was assed by western blot, qRT-PCR, wound-healing assay, matrigel
assay.
Results: Flow cytomety analysis showed that histone acetylation reduce the expression of endothelial
markers such as CD31, CD105, CD117, CD133, CD144, VEGFR2. Furthermore histone acetylation
inhibited neovascularization in vitro, acting in the processes of proliferation, adherence, migration and
in the formation of vascular network structures.
In conclusion: discovering patterns of acetylation involved in the differentiation of stem cells to
different cell types, open new opportunities at the interface between chemistry and stem cell biology,
and can improve applications of stem cells in tissue engineering and regenerative medicine.
A.19 - The haptoglobin 1-2 polymorphism is not related to peripheral arterial disease
R Jahrbacher, EA Marx-Neuhold, B Zulus, W Renner.
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, 8036
Graz, Austria.
Background: Haptoglobin is an acute phase plasma protein and synthesized primarily in the liver and
secreted into the bloodstream. The molecule is a potent antioxidant with the main function of
scavenging free hemoglobin, which is toxic to cells. Furthermore , direct angiogenic, antiinflammatory, and immunomodulatory properties of haptoglobin have also been reported, suggesting a
potential role in cardiovascular disease. Haptoglobin consists of two chains, the alpha-chain and the
beta-chain, deriving from a single polypeptide after proteolytic cleavage. The alpha-chain exists in two
major forms, the alpha-1-chain and the longer alpha-2-chain, which contains a direct repeat of 63
amino acids. This structural heterogeneity is the result of an intragenic duplication of about 1.7 kb
within the Haptoglobin gene (HP). The haptoglobin polymorphism leads to three principal genotypes,
HP 1-1, 1-2, and 2-2, which have biologically important differences in their antioxidant, scavenging,
and immunomodulatory properties. The role of these genotypes for peripheral arterial disease (APD)
remains unclear.
Objective: We investigated the potential association of haptoglobin genotypes with PAD.
Design: DNA samples of 846 patients with documented PAD and 734 control subjects were
genotyped for the haptoglobin 1-2 polymorphism.
Results: Haptoglobin genotype frequencies were not significantly different between PAD patients (11: 42.3%, 1-2: 42.7%, 2-2: 15.0%) and control subjects (41.6%; 46.6%; 11.9%, p = 0.12). The
haptoglobin polymorphism was furthermore not associated with age at first onset of PAD, anklebrachial-index, or PAD stage.
Conclusions: The Haptoglobin 1-2 polymorphism is not associated with PAD.
A.20 - Polymorphism in BIRC5 gene is associated with DAS28 in Rheumatoid arthritis patients
B. Jenko1, S. Praprotnik2, D. Logar2, M. Tomsic2, V. Dolzan1
1
Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia,
University Medical Centre Ljubljana, Ljubljana, Slovenia
2
Objective: Survivin, a member of the inhibitor of apoptosis (IAP) family is associated with
unfavorable and erosive course of disease in rheumatoid arthritis (RA) patients. It was recently shown
that survivin’s expression levels may be modified by polymorphisms in BIRC5 promoter region. We
investigated the association between BIRC5 promoter polymorphisms, survivin plasma levels and
disease activity in RA patients.
Design: The study included a testing group of 123 patients and validation group of 150 patients,
initially treated with low dose methotrexate (MTX) monotherapy. The data on gender, age, disease and
treatment duration, MTX dose, presence of bone erosions, RF or anti-CCP seropositvity and DAS28
were obtained from the medical records. BIRC5 -31G>C, -241C>T and -625G>C polymorphisms
were genotyped using allele-specific real time polymerase chain reaction (KASPar, Kbiosciences,
Hoddesdon, UK). Survivin levels were measured using a sandwich enzyme-linked immunosorbent
assay (Human Survivin ELISA Kit, Boster Biological Tehnology Co., Inc.).
Results: Survivin positivity in plasma was not associated with BIRC5 polymorphisms or DAS28.
BIRC5 -31G>C was significantly associated with DAS28 after 6 months of treatment (p=0.036). The
association was still present at the time of inclusion in the study in testing (p=0.052) group as well as
marginally associated in validation group (p=0.057), at this time point the disease duration was 3 and
5.6 years, respectively. In both groups BIRC5 -31G>C was also significantly associated with disease
activity at inclusion (p<0.000 and p=0.018, respectively).
Conclusions: BIRC5 polymorphisms were not associated with survivin plasma levels, however BIRC5
-31G>C may influence disease activity in RA patients.
A.21 - Study of polymorphisms rs2476601, rs3761847, rs660895 and rs7574865 involved in
clinical and immunological manifestations of rheumatoid arthritis in 472 individuals
I. Karanika and N. Drakoulis
National and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy.
Background and objectives: Rheumatoid Arthritis is a multifactorial disease, the possible genetic
components of which are continuously and systematically researched. The discovery of SNPs (Single
Nucleotide Polymorphism,) by the addition, deletion or change of a base have led to the discovery of
new genetic correlations with the disease. The objective of this particular study is to demonstrate how
the polymorphisms in specific loci RS2476601, RS3761847, RS660895 and RS7574865 affect various
clinical and immunological manifestations of RA.
Methods: The analysis was carried out on a sample of 472 healthy individuals from Greece. Genomic
DNA used was isolated from the epithelial cells of the oral cavity and analyzed by Real-Time
Polymerase Chain Reaction.
Results: The genotype of GA RS660895 is 17.37% of the sample, while the GG is the 2.12%. The
RS3761847 occurring in 11, 44% as GG genotype and 44.91% as GA .While RS2476601 is 8.26%
having a GA genotype and 0.21% having an AA. The homozygous RS7574865 as TT is 6.57%, while
the GT is 39.40%.
Conclusion: All 4 polymorphisms were found in the Greek population, however, RS3761847
alongside RS7574865 show higher MAF (Minor Allele Frequency) and may be responsible for
increased susceptibility to RA.
References: Karanika I., Drakoulis N. Analysis of polymorphisms RS2476601, RS3761847,
RS660895 and RS7574865 involved in clinical and immunological manifestations of rheumatoid
arthritis. In: N. Drakoulis (ed.) Contribution of Genetic Analyses in Predisposition, Diagnosis,
Prognosis and Therapeutic Approach to Disease, Chapter 5, I. Levakos, N. Papaioannou (eds.)
Orthopedics, sports medicine. APR-Publicartions, Athens 2013, pp 224-234.
A.22 - Effect of a 3-month intervention with Corinthian Raisins in Greek males and females
with non alcoholic fatty liver disease; a mid-term evaluation
AC Kaliora1, M Stoupaki1, A Gioxari1, PT Kanellos1, A Kokkinos2, GVZ Dedoussis1, VT Karathanos1,
S Ladas2
1
Department of Science of Dietetics-Nutrition, School of Health, Science and Education, Harokopio
University, Athens, Greece
2
First Department of Propaedeutic and Internal Medicine, Laiko General Hospital, National and
Kapodistrian University, Medical School, Athens, Greece
Our objective was to investigate the effects of dietary intervention with Corinthian Raisins in
comparison with commonly given recommendations for therapeutic lifestyle changes subjects with
nonalcoholic fatty liver disease (NAFLD). This is a 3-month intervention study including 60 males
and females with NAFLD, matched for age and body mass index (BMI). Subjects were randomized
into two groups to receive 36g of CR daily (n=30) or control (n=30), both counseling for therapeutic
lifestyle changes. Ultrasound scanning and shear-wave elastography were applied as diagnostic tools
at baseline. Medical history, clinical and anthropometric indices, physical activity data and
biochemical markers, were collected for all the participants at baseline, and after 3 months. Several
anthropometric and clinical measurements were regulated in patients enrolled the intervention, rather
than the control group. Blood glucose reduced significantly in the intervention but not in control
group, along with systolic and diastolic blood pressure. This is an interim report of a 6-month
intervention being in progress within the National Strategic reference Framework (NSRF) 2007-2013
Programme for Development-Quality of Life for Everyone, European Regional Development Fund
(ERDF) 2007-2013 OPCE II: Operational Programme “Competitiveness and Entrepreneurship”
Hellenic Ministry of Development and Competitiveness (Grant No:09-ΣΥΝ-12-890)
A.23 - Integration of proteomic data and clinical annotations reveals ciliopathy mechanisms.
K. Koutroumpas1, T.J.P. van Dam2, G. Toedt3, Q. Lu4, J. van Reeuwijk5, K. Boldt6, M. Ueffing6, T.
Gibson3, R. Russell4, R. Roepman5, M. Huynen2, M. Elati1, F. Képès1.
1. Institute of Systems and Synthetic Biology, Genopole, CNRS, Université d'Evry, Evry, 91030,
France, 2. Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre,
Nijmegen 6500 HB, The Netherlands, 3. European Molecular Biology Laboratory, Meyerhofstraße 1,
69012 Heidelberg, Germany, 4. BioQuant, University of Heidelberg, 69120 Heidelberg, Germany, 5.
Department of Human Genetics, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10,
6525, GA, Nijmegen, The Netherlands 6. Institute for Ophthalmic Research, Centre for
Ophthalmology, University of Tübingen, Roentgenweg 11, 72076, Tübingen, Germany
Background: Understanding the molecular and cellular mechanisms of diseases is vital for dissecting
diseases pathogenesis, identifying appropriate therapeutic targets and designing effective treatments.
Recent advances in DNA sequencing technology have provided a torrent of genetic data that can now
be used to elucidate the genetic basis of human diseases. A concensus has emerged among biologists
that to fully exploit the available data, they have to be correlated with additional research. This is
especially important for rare-disease genetics, mainly because of the small number of available
patients.
Objective: Computational methods could be employed to tackle the problem of limited genetic data.
Integration of diverse biological data could provide additional evidence to support experimental
observations and in silico observations can be used for the design of new experiments.
Design: A statistical method that predicts disease causing genes by integrating protein interaction data
and clinical phenotype annotations has been developed. For a given protein in the network the method
predicts the clinical phenotypes that may appear upon protein alteration based on the phenotypes
associated with the neighbours of the protein.
Results : Application to protein interaction data from the SYSCILIA project (http://syscilia.org) and
phenotype annotations from the HPO project (http://www.human-phenotype-ontology.org) succesfully
predicts candidate Nephronophthisis genes. The results also imply that abnormal Hh signaling may be
the cause of Nephronophthisis and GPCR mislocalization a possible way by which cilia defects affect
Hh signaling.
Conclusions: The results indicate that the developed method can be useful for the dissection of
disease pathogenesis by predicting disease genes and drawing new hypoteses on the underlying
mechanisms. Such hypotheses can assist in the design of new targeted experiments.
Acknowledgements: Funded by EU FP7 under GA nr. 241955, project SYSCILIA.
A.24 - Prediction of candidate ciliopathies by phenotypic similarity.
K. Koutroumpas, M. Elati, F. Képès.
Institute of Systems and Synthetic Biology, Genopole, CNRS, Université d'Evry, Evry, 91030, France
Background: Ciliopathies comprise a group of diseases associated with genetic mutations encoding
defective proteins that affect the structure or function of cilia. As cilia are a component of almost all
cells, ciliary defects have been linked to a constellation of phenotypes (clinical features) that include:
retinal degeneration, brain malformations, skeletal abnormalities, and renal disease. Ciliopathic
phenotypes have been associated with mutations in over 40 genes to date.
Objective: With the ciliary proteome being composed of around 1000 proteins, it is likely that several
other disorders, associated with ciliary phenotypes, will be ascribed to mutations in ciliary genes. We
aim at the prediction of such candidate ciliopathies based on their clinical similarity with known
ciliopathies.
Design: For the identification of novel ciliopathies we employed phenotype annotations from the
Human Phenotype Ontology (HPO) project (1). A semantic similarity measure is used to access the
similarity of a pair of phenotypes in HPO. Phenotypic similarity between two diseases is then
calculated based on the similarity of the HPO phenotypes associated with the diseases. We computed
phenotypic similarity between all diseases listed in the Online Mendelian Inheritance in Man (OMIM)
database (http://www.omim.org) and a super-ciliopathy, i.e. a disease associated with all known ciliary
phenotypes.
Results: As expected, known ciliopathies have a high phenotypic similarity with the super-ciliopathy,
providing an intuitive validation of the methodology. Moreover, several non-ciliopathy diseases
appear to be similar to the super-ciliopathy, as they are associated with ciliary phenotypes. Such
diseases could be candidate ciliopathies. For some of them recent data indicate possible connections
with ciliary function, while for others it is known to be caused by dysfunction of biological processes
affected by cilia.
Conclusions: Our study shows that phenotypic similarity can be useful in the prediction of new
ciliopathies. In some cases novel ciliopathic genes are predicted and hypotheses about disease
pathogenesis are drawn. The methodology can also be used for patient diagnosis, which is especially
challenging because of the extended genetic and phenotypic overlap of ciliopathies.
Acknowledgements: Funded by EU FP7 under GA nr. 241955, project SYSCILIA.
Reference: Robinson PN, Kohler S, Bauer S, Seelow D, Horn D, Mundlos S. The Human Phenotype
Ontology: a tool for annotating and analyzing human hereditary disease. Am J Hum Genet. 2008
Nov;83(5):610-5.
A.25 - AMPK Epidermal Inhibition Promotes HuR Cytoplasmic Localization Eliciting Posttranscriptional Inflammatory Response in Psoriasis
L. Le Gallic
School of Medicine, University of Montpellier-Nîmes IBMM UMR 5247 CNRS, Nîmes, France
Most molecular mechanisms underlying the establishment of psoriasis, a chronic skin inflammation,
remain to be elucidated. Here, we show that the RNA binding protein HuR re-localizes in
keratinocytes cytoplasm of psoriatic patients suggesting implication in the stabilization of numerous
transcripts, as we demonstrate for the IL-20 cytokine. We characterize, by ribonucleoprotein
immunoprecipitations analyzed by high throughput sequencing, HuR RNA targets and identify that
numerous psoriasis up-regulated transcripts are regulated by HuR, supporting an implication of HuR
in pathophysiological processes such as morphological, immune and metabolic inflammatory
responses. We finally identify the metabolic sensor AMPK as being responsible for HuR cytoplasmic
re-localization since its activity is severely impaired in human psoriatic epidermis and since in vivo
drug-mediated AMPK inhibition in mouse epidermis promotes HuR cytoplasmic localization, IL-20
over-production as well as psoriasis–like histological changes. These results provide insights into
molecular links between metabolism and post-transcriptional networks during chronic inflammation.
Highlights
• IL-20 and IL-22 up-regulation in psoriasis are post-transcriptional
• p38 is not activated in psoriatic keratinocytes while HuR re-localizes in cytoplasm
• Numerous psoriatic up-regulated transcripts are HuR mRNA targets
• AMPK inhibition promotes HuR cytosolic localization and IL-20 over-production
A.26 - Prediction of Methotrexate Therapeutic Outcome in Rheumatoid Arthritis Portuguese
patients: Candidate Genes encoding Methotrexate Membrane Transport Proteins
A. Lima1,2,3, M. Bernardes4, H. Sousa2, R. Azevedo2, R. Medeiros2,3,5, V. Seabra1
1
CESPU, Department of Pharmaceutical Sciences, ISCS-N.
Molecular Oncology Group CI, IPO-Porto.
3
Abel Salazar Institute for the Biomedical Sciences, UP.
4
Rheumatology Department of CHSJ. 5Research Department-Portuguese LPCC-N.
2
Background: Methotrexate (MTX) is used for rheumatoid arthritis (RA). MTX membrane transport
proteins (solute carriers–SLCs and ATP-binding cassette–ABCs) can be major determinants of
pharmacokinetics, adverse drug reactions (ADRs) and clinical response profiles.
Objective: Evaluate the influence of SNPs in genes encoding for MTX membrane transport proteins
in MTX therapeutic outcome in Portuguese RA patients.
Design: Clinicopathological data from 233 RA patients treated with MTX were collected, outcomes
(non-response–MTX-NonResp; overall toxicity–MTX-Tox; and gastrointestinal toxicity–MTXGastroTox) were defined, and patients were genotyped for 23 SNPs. Genotype and haplotype analyses
were performed using multivariate methods. Genetic risk indexes for toxicity and non-response (TRI
and GRI) were created.
Results: Increased risk for MTX-NonResp was associated to SLC16A7 rs3763980 A carriers
(P=0.050), SLC22A11 rs11231809 T carriers (P=0.021), ABCC1 rs246240 G carriers (P=0.009) and
rs3784864 G carriers (P=0.034). Estimated haplotypes weren’t associated with MTX-NonResp.
Increased GRI wasn’t associated with increased risk for MTX-NonResp. Increased risk for MTX-Tox
was associated to SLC19A1 rs7499 G carriers (P=0.017), SLC46A1 rs2239907 GG (P=0.028),
and SLCO1B1 rs4149056 T carriers and TT (P=0.037 and P=0.018). Estimated haplotypes weren’t
associated with MTX-Tox. Increased TRI was associated with increased incidence of ADRs
(P=0.020). Index 3 were 18.42 times more likely to present an ADR when compared to index 1
(P=0.001). Increased risk for MTX-GastroTox was associated to SLC19A1 rs7499 G carriers and GG
(P=0.013 and P=0.035), SLC19A1 rs1051266 G carriers (P=0.039), SLCO1B1 rs4149056 T carriers
and TT (P=0.039 and P=0.024) and ABCG2 rs13120400 C carriers (P=0.042). GGAG haplotype for
SLC19A1 rs7499, rs1051266, rs2838956 and rs3788200, was associated with MTX-GastroTox when
compared to AAGA haplotype (P=0.029). Increased TRI for MTX-GastroTox was associated with
increased incidence of gastrointestinal disorders (P=0.009). Index 4 were 22.06 times more likely to
present MTX-GastroTox when compared to index 1 (P=0.007).
Conclusions: This study revealed that SNPs in SLCs and ABCs may be helpful to elucidate which
patients will not benefit from MTX treatment. The proposed TRI and GRI highlight the importance of
genotyping patients and the urgency of developing the field of personalized medicine for the
prediction of MTX therapeutic outcome.
A.27 - A set of applications to understand protein-protein interaction networks and the impact
of mutations
Q. Lu1, M. Betts1, Y. Jiang1, R. Russell1
1. BioQuant, University of Heidelberg, 69120 Heidelberg, Germany
Background: Current sequencing and proteomics techniques identify thousands of protein mutations
or posttranslational modifications in a single experiment, creating an acute need to understand how
they affect biological systems. Systematic interrogation of mutation or protein modification data is
important to identify sites with functional consequences and to deduce global consequences from
entire datasets.
Objective: Bearing the complexity of the genomic/proteomic data in mind, we aim to develop an
integrated analysis system employing the current knowledge combining computational methods like
protein 3D alignment and statistical modeling. This system should enable modeling protein amino acid
changes on biological systems, analysing mutation/variation and PTM datasets to assess their impact
integrated on the interactome, and developing a method to predict the impact of protein variations on
biological systems.
Design: We have developed an integrated system composed of new developed and existing
applications to perform evaluation of known interactions (UNINT), analysis of mutation/variation and
PTM dataset to assess their impact on the interactome (MECHISMO), on the level of protein domains
(DOMNET), and between protein and linear motifs (DILIMOT).
Results: This integrated system enables simultaneous consideration of thousands of three-dimensional
structures of proteins, chemicals and nucleic acids integrated with the interactome to predict
mechanistic consequences for mutation and modification data, including whether sites enhance or
diminish interactions. Analysis of cancer sequencing, proteomics and disease mutation data shows that
MECHISMO can suggest novel insights both for individual changes and entire datasets. DOMNET
and DILIMOT generate overviews of the domain-domain and domain-motif network helping to lock
candidates for further studies.
Conclusions: Our system provides integrated analysis covering multiple aspects of protein-protein
interaction mechanism. The web interface balances the complexity of problem and simplicity of use.
Results of several studies indicate that it helps dissecting diseases by understanding the whole picture
or just cherry picking for disease causing candidates.
Acknowledgements: Funded by DFG Excellence Cluster ‘CellNetworks’ and EU FP7 under GA nr.
241955, project SYSCILIA
A.28 - Genetic polymorphism of the serotonin receptor HTR2C-759C>T in schizophrenia
patients undergoing treatment with olanzapine in a north indian population
S. Maity1, V. Subbiah 1, M. Munisamy1, R. Sagar.2
1
Department of Neurobiochemistry, All India Institute of Medical Sciences, Delhi, India
Department of Psychiatry, All India Institute of Medical Sciences, Delhi, India
2
Background: Schizophrenia is a devastating psychiatric syndrome, which affects about one percent of
people world-wide. The symptoms of schizophrenia usually appear at young age, by the second and
third decades of life.
Objective: To investigate the association between the genetic polymorphism of serotonin receptor
HTR2C-759C>T and the serum concentration of Olanzapine in responders and non-responders of the
Schizophrenia patients.
Design: 100 unrelated chronic schizophrenia patients (68 male and 32 female; mean age: 52.8±11.6
years; mean duration of illness: 6.1±9.3 years; mean age of onset: 26.8±8.1 years) were enrolled in our
study from the Department of Psychiatry, AIIMS, New Delhi. Diagnosis of schizophrenia patients was
according to Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria with consensus
based on the clinical interviews and case records. All patients were of North Indian origin. All patients
received Olanzapine at an initial dose of 5 mg/day, and dosage was gradually increased to 15mg/day
during the first week. Serum concentrations of Olanzapine were measured using a specific HPLC
assay. We assessed the response of positive and negative symptoms by PANSS at the start of
treatment and after eight weeks. Patients were considered as responders if their PANSS score
decreased at least 30% responder (n=50) and non-responders if it not (n=50). PCR-RFLP technique
was used for genotyping.
Results: Significant differences were seen in the genotype distribution (χ2 =5.84, d.f. = 2, p =0.01)
and allele frequencies (χ2 =7.62, d.f. = 1, p =0.005; odds ratio = 0.13 95% confidence interval = 0.240.79) between the responders and non-responders. In this study, the mean olanzapine serum levels in
responders was found to be 36.2 ± 18.0 ng/ml and in non responders, 51.8 ± 29.7 ng/ml receiving an
olanzapine dose of 15 mg/day.
Conclusions: HTR2C-759C>T polymorphism was seen to be in Hardy–Weinberg equilibrium (P >
0.01) and showed significant genotypic as well as allelic association with responders Vs non
responders to olanzapine (P<0.05). Our preliminary results suggest that genetic polymorphism of
HTR2C-759C>T do influence the metabolism and therapeutic outcome of Olanzapine in
schizophrenic patients.
Acknowledgements: Funding for this research was supported by Indian Council of Medical Research
(ICMR), New Delhi.
Reference: 1.Akimoto, T.; Morokawa, Y.; Kumai, T.; Sekiguchi, G.; Nakaya, S.; Suzuki, H.; Gen, K.;
Kobayashi, S.; Aoba, A.Polymorphism of serotonin and histamine receptors and clinical efficacy of
olanzapine. International Clinical Psychopharmacology: May 2004 - Volume 19 - Issue 3-pp 177-178 .
A.29 - Genetic Susceptibility Biomarkers of Alzheimer’s Disease Development: The Role of
rs7412 and rs429358 Polymorphisms
M. Μalliou1,2 N. Drakoulis2
1
University of Perugia Faculty of Pharmacy, Department of Pharmacology, 2National and Kapodistrian
University of Athens, School of Health Sciences, Faculty of Pharmacy
Background & Objectives: Alzheimer's disease is a degenerative disease of the central nervous
system characterized by loss of memory, difficulty in executing daily tasks, anxiety, aggression and at
the final stages the patient falls in coma and death occurs. In this study the polymorphisms rs7412 and
rs429358of apolipoprotein E (ApoE) gene have been analyzed, to reveal their relationship with
Alzheimer’s disease, separately and combined on the same allele.
Methods: DNA from 95 cases, with confirmed AD diagnosis, and 100 non affected controls, was
isolated by standard DNA extraction and real time Polymerase Chain Reaction was applied to detect
the polymorphisms in all subjects. Statistical analysis was performed and in order to determinate
possible genetic disorder that predisposes to AD the odds ratios (OR) and 95% confidence intervals
(95%CI) of each polymorphism between cases and controls were evaluated. Further, comparison
between male and female within the AD patients was carried out, to reveal differences in the
susceptibility between genders.
RESULTS: In this study OR of polymorphism rs7412, alone, in the Alzheimer population, in male and
in female, were 0.69, 0.61 and 0.85 respectively, as compared to age matched controls. The OR
rs429358 polymorphism, alone, in the Alzheimer group, in male and in female patients were 3.78,
2.91 and 5.90, respectively, as compared to aged matched controls.
Conclusion: The contribution of ApoE in the development of AD was confirmed with the exception
of the polymorphism rs7412 when not combined with other polymorphisms. The polymorphism
rs429358 alone or in combination with rs7412 on the same allele predisposes male to Alzheimer’s
disease twice as much as compared to females.
References: Malliou M., Drakoulis N. The Prevalence of Polymorphisms rs7412 and rs429358 of
ApoE Gene Associated with Alzheimer’s disease, in a Sample of Greek Patients and Controls. In: N.
Drakoulis (ed.) Contribution of Genetic Analyses in Predisposition, Diagnosis, Prognosis and
Therapeutic Approach to Disease, Chapter 4, K. Koumakis, C. Touloumis (eds.) Central nervous
system. APR-Publications, Athens 2013, pp 167-176.
A.30 - How do genes ınfluence personalıty: a study ın 830 greek volunteers
G. Marinos, N. Naziris and N. Drakoulis
National and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy.
Background & Objectives: Intelligence consists of a series of competencies (e.g. perception,
learning, adaptation). Decision making is defined as the conscious choice between given options,
relating to a problem. The genetic background and the environment are the key elements for the
personality characteristics of the human being. The object of this study is to determine the rs324420,
rs1800497, rs363050, rs6265, rs1328674 gene effect on these topics, in 830 Greek Subjects.
Methods: The population of the volunteers is described, based on genotype, sex, with the respective
frequencies, including the Minor Allele Frequency (MAF). A potential relationship of the volunteer
genus with the above characteristics is checked and finally, volunteers are classified; a volunteer
receives +1/-1, for each genotype and haplotype, which enhances/relegates his intelligence or his
decision-making respectively.
Results: No statistically significant gender-characteristics correlation is observed. At a rate of 92.5%,
the volunteers are characterized by prudence and temperance of thought, with only a small proportion
of them (7.5%) being genetically spontaneous and adventurous. Regarding intelligence, the population
is around an average and a little above it, at a rate of 96.3%, while the edges of the scale suggest only a
0.5% of the volunteers, who, although the “smartest”, somehow lack prudence.
Conclusion: Individuals with low cognitive ability may be more prudent than others and vice versa,
while the “smartest” ones tend to be more risky, in decision-making. Therefore, intelligence and
decision-making, after all, may be less linked to each other than expected. These results may be useful
in targeted and personalized therapies of relevant diseases.
References: Marinos G., Naziris N., Drakoulis N. Genes and Personality Characteristics: Study of the
Effect of the Genetic Background on Intelligence and Decision Making in 830 Greeks. In: N.
Drakoulis (ed.) Contribution of Genetic Analyses in Predisposition, Diagnosis, Prognosis and
Therapeutic Approach to Disease, Chapter 4, K. Koumakis, C. Touloumis (eds.) Central nervous
system. APR-Publicartions, Athens 2013, pp 195-206.
A.31 - Rapid TaqMan:“Fast and reliable genotyping using whole blood without DNA
purification“.
EA Marx-Neuhold, R Jahrbacher, B Zulus, W Renner.
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, 8036
Graz, Austria.
Background: Genotyping methods using fluorescent probes, such as TaqMan assays, usually require
time-consuming purification of DNA from blood.
Objective: We developed an optimized protocol for rapid genotyping of Factor V Leiden and the
prothrombin 20210GA>A mutation using TaqMan probes in whole blood („Rapid TaqMan“).
Design: We performed a standard TaqMan reaction using diluted EDTA blood instead of DNA.
Briefly, whole blood was diluted with water, mixed on a vortex shaker and centrifuged. In a stepwise
procedure we optimized the (1) the dilution factor, (2) the mixing time, (3) the amount of diluted
blood per reaction, and (4) the temperature profile of the PCR program. After optimization of the
protocol, thirty samples were genotyped for Factor V Leiden and the prothrombin 20210GA>A
mutation with the Rapid TaqMan protocol as well as a standard TaqMan protocol using purified DNA.
Results: The Rapid TaqMan protocol using whole blood delivered unambigous and clear results for all
samples. No genotype discrepancies between the Rapid TaqMan protocol and the standard TaqMan
protocol using purified DNA were observed.
Conclusions: The rapid TaqMan protocol allows genotyping of whole blood samples without DNA
purification..
A.32 - Nation-wide genetic screening for patients with bilateral nonsyndromic sensorineural
hearing loss (SNHL) in Slovakia.
I. Mašindová1, L. Varga1,2, M. Hučková1,4, M. Balogová1, Ľ. Šuchová 3, M. Profant2,
D. Gašperíková1,4 and I. Klimeš1,4
1
Diabgene Laboratory, Institute of Experimental Endocrinology, Slovak Academy of Sciences,
Bratislava, Slovakia
2 st
1 Otorhinolaryngology Clinic, Faculty of Medicine and University Hospital, Comenius University in
Bratislava, Slovakia
3
Department of Phoniatry, University Hospital in Bratislava, Slovakia
4
Centre for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia
Introduction: Etiology of hearing impairment is very heterogeneous. Approximately 50% of all deaf
born cases are attributed to genetic causes. Exact data about genetic etiology of bilateral sensorineural
hearing loss in our country are not fully available.
Therefore, the aim of our study was to carry out the genetic screening for selected genes in patients
with bilateral nonsyndromic SNHL throughout Slovakia.
Patients and Methods: Since 2010 we have recruited 708 subjects from all boarding schools for
children with hearing disabilities in Slovakia and University Hospital in Bratislava. For genetic
testing, 505 unrelated individuals with clinically manifested bilateral SNHL were selected and
sequenced for GJB2 gene. In 286 patients, we have also carried out analyses of GJB6, GJB3 and
POU3F4 genes using the MLPA methodology.
Results: We identified 17 already known mutations, eight polymorphisms and three variants in the
GJB2 gene. Homozygous and compound heterozygous mutations were present in 20% and 11% of the
subjects, respectively. Proportion of the subjects without having any pathogenic allele, reached 61%.
In one individual, a simultaneous occurrence of the 309 kb large deletion in GJB6 with the GJB2
mutation was identified. No mutations were found in other genes investigated.
Conclusions: 1. We have established the mutation profile of GJB2/GJB6 genes in subjects identified
across whole Slovakia, and 2. we have confirmed the genetic etiology of hearing impairment in 31%
of individuals studied. 3. Our findings serve as key information for genetic counseling and clinical
prognosis of SNHL.
Supported by: APVV/0148-10, “KCMM ITMS 26240220071”
A.33 - Epistasis in Blood Pressure Regulation
NC Ndiaye1, S EL Shamieh1, MG Stathopoulou1, G Siest1, MY Tsai2, A Benetos3, HA Murray4, JV
Lamont4, P Fitzgerald4, S Visvikis-Siest1
UMR INSERM U1122; IGE-PCV ‘Interactions Gène-Environnement en Physiopathologie
CardioVasculaire’, Université de Lorraine, Nancy, France,
2
Department of Laboratory Medicine & Pathology, University of Minnesota, Minneapolis, MN,
55455-0392, USA,
3
INSERM U1116, ‘Hypercoagulabilité, rigidité et vieillissement artériels’, Université de Lorraine,
Nancy, France,
4
Randox Laboratories Ltd, Crumlin, Antrim, United Kingdom
1
Background: Classic candidate gene and genome-wide association studies failed to appropriately
elucidate genetic mechanisms underpinning blood pressure regulation. Indeed, interaction
investigations, on gene-gene/epistatic synergies for instance, are frequently neglected whereas those
mechanisms could partially explain the large missing heritability and discrepancies observed in blood
pressure-related studies.
Objective: Our aim was to identify epistatic interactions associated with blood pressure levels.
Design: We performed a pre-planned two-phase investigation on a panel of genetic variants associated
to various metabolic pathways involved in cardiovascular risk. The significant epistatic interactions
regulating blood pressure observed in a discovery phase 1 gathering unrelated French adults were
further investigated in a replication phase 2 gathering independent samples.
Results: Among the significant [single nucleotide polymorphism (SNP) – blood pressure] associations
observed, some may act through altering the corresponding protein levels: SNPs rs5742910 and
rs6046 in F7 and rs1800469 in TGFB1; whereas some may be functional through altering the
corresponding protein structure: rs1800590 in LPL and rs2228570 in VDR. Three epistatic
interactions: VCAM1 (rs1041163) * APOB (rs1367117), SCGB1A1 (rs3741240) * LPL (rs1800590),
and F7 (rs6046) * SELE (rs5355) were involved in blood pressure regulation and in silico analyses
yielded putative functional properties of the SNPs involved through the alteration of corresponding
protein structures. Furthermore, rs6046 in F7 was positively associated with NAMPT mRNA levels
expressed in peripheral blood mononuclear cells, suggesting a possible involvement of NAMPT
expression in blood pressure regulation. Confirming this hypothesis, further transcriptomic analyses
showed that increased NAMPT mRNA levels were positively correlated with ICAM1, SELL, FPR1,
DEFA1-3, and LL-37 genes expression.
Conclusions: Blood pressure genetic epidemiology lies in the investigation of synergistic
mechanisms, i.e. epistasis, involving various metabolic pathways in order to highlight novel
pathophysiological targets.
A.34 - Orientation towards health and individuals in 20th century medical projects – a historical
insight
D. Olivier (IHPST, Université Paris 1 Panthéon-Sorbonne, Paris, France.
Supervisor : Pr J. Gayon, IHPST, Paris, France
Often described as a technology-driven science, systems medicine is moving fast.
Objectives: We will offer a historical insight into predictive medicine projects, in order to show that
this « new » medicine is the result of a 60-year old medical project. The methodology is that of a
historical enquiry into mid-20th century predictive medicine and prospective biology projects. We
argue that looking at the past is a considerable help to understand the vision which lies at the core of
systems medicine.
Results: We will present three main characteristics of these early projects of predictive medicine.
First, the essential role of technology and automation has to be acknowledged. This may seem obvious
to anyone concerned with systems medicine and genomics. However, we have to insist on the fact that
automation began before genome sequencing. Laboratory medicine played a crucial role in the
development of these projects, and keeping that in mind will help us understand why systems
medicine cannot be reduced to the exploration of the genome. Second, early predictive medicine
projects are linked to an orientation towards individuals. Not only are individuals the privileged target
of this medicine – instead of populations – but predictive medicine also aims at a direct knowledge of
the individual physiology. Pharmacogenomics, P4 medicine, echo the late 1960s projects of predictive
medicine and the fierce defence of the exploration of “biological individuality” by Roger Williams for
instance. This focus on individuals also helps to understand the importance of biological variations in
these approaches. Third, mid-sixties projects reveal a strong orientation towards health: medicine is
viewed as a science of health instead of a science of disease. Early predictive medicine projects, such
as Emmanuel Cheraskin’s one, are explicitly health-oriented. Recently, P4 medicine (especially when
it comes to the “predictive”) has raised a number of fears: would medicine become capable of
revealing the impending pathological fate of each individual? Historical insight helps to balance this
view by showing that a new knowledge of health was at the core of predictive medicine projects.
Conclusion: Therefore, prospective biology and medicine has a history which is worth exploring in
order to keep in mind the core elements of the vision proposed: automation and technologies are meant
to serve the development of a new science of individual health.
A.35 - Genetic variability in CYP2E1 and Catalase gene and alcohol dependence in Slovenian
acutely alcohol dependent patients, abstainers and healthy controls
A Plemenitas2, M Kastelic1, S Porcelli3, A Serretti3, B Kores Plesnicar2, V Dolžan1
1
Institute of Biochemistry, Faculty of Medicine, Ljubljana, Slovenia;
Psychiatric Clinic Ljubljana, Ljubljana, Slovenia;
3
Institute of Psychiatry, University of Bologna, Bologna, Italy.
2
Background: Heritability has an important role in the development of alcohol dependence. Genes
involved in ethanol metabolism, including CYP2E1 and catalase, may determine variability in
response to alcohol.
Objective: The present study explored whether specific single- nucleotide polymorphisms in alcohol
metabolic pathway CYP2E1 -1053C>T (rs2031920) and CAT -262C>T (rs17602729) could be
associated with alcohol dependence or alcohol related psychopathological symptoms.
Design: Male Caucasian subjects were included: 101 acutely alcohol dependent inpatients, 100 former
alcohol-dependent patients and 97 healthy blood donors. The following questionnaires were
employed: AUDIT, Zung Depression and Anxiety scale, Brief Phobia Scale, Yale-Brown Obsessive
Compulsive Scale and Obsessive Compulsive Drinking Scale and Buss-Durkee Hostility Inventory.
Subjects were genotyped for CYP2E1 -1053C>T and CAT -262C>T.
Results: Differences in distribution of CAT genotypes (p = 0.004) and alleles (p = 0.001) were
observed across the three groups. We observed higher frequency of CAT -262T alleles in alcohol
dependent patients (OR = 1.74, 95% CI = 1.164 - 2.610). Among acutely dependent patients, carriers
of CAT-262T allele showed a trend of obtaining higher scores on AUDIT questionnaire (p = 0.062). In
the same group of acutely dependent patients CYP2E1-1053C>T alleles were significantly associated
with Yale-Brown Obsession subscale score (C allele mean score ± SD = 4.1 ± 4.4 and T allele mean
score ± SD = 8.3±4.7, p = 0.005) as well as with Zung Anxiety Scale score (C allele mean score ± SD
= 34.8 ± 8.5 and T allele mean score ± SD = 42.2 ± 9.7, p = 0.011).
Conclusions: This is the first study investigating the distribution and influence of genetic variants
within genes involved in alcohol metabolism in Slovenian alcohol dependent subjects and controls.
Our findings suggest that the CAT -262C>T genetic polymorphism influences the susceptibility to
alcohol dependence and severity of alcohol dependence, while CYP2E1 -1053C>T polymorphism
influences the expression of obsessive-compulsive and anxiety symptoms possibly related to oxidative
stress.
Acknowledgements: The study was financially supported by the Ministry of Education, Science, and
Sport of the Republic of Slovenia.
A.36 - Effect of dıhydrotestosteron on adult stem cells derıved from human post-natal cord
blood and matrıx
M. A. Popa*, M. C. Corotchi*, M. Simionescu (*, equal contribution)
1
Angiogenesis and Cardiovascular Remodeling Group, Department of Regenerative Medicine,
Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy,
Bucharest, Romania
Introduction Human mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) have
been established as valuable candidates for cardiovascular remodeling. Based on the facts that adult
MSC and EPC have the potential to repair/regenerate tissue following cardiac injury, and that
androgens (i.e. dihydrotestosterone – DHT) influence cell growth and function, we hypothesize that
DHT may have a positive effect on the regenerative capability of adult stem /progenitor cells.
Materials and Methods EPC were isolated from the umbilical cord blood and MSC from Wharton’s
jelly. Morphological and functional characterization of both populations was made according to “gold
standard” protocols. The presence of androgen receptors (AR), the proliferation rate and the
quantification of the pro-angiogenic genes were assessed using immunofluorescence, cell impedance
measurements and RT-PCR. Using a Real Time Cell Analyzer with impedance measurements, we
tested the cell proliferation rate at different concentrations of DHT (1 nM, 10 nM and 100 nM) and at
different exposure time (48h, 80h and 96h) .
Results and Discussions. Employing fluorescent anti - AR antibody we found that both, isolated EPC
and MSC expressed androgen receptors (AR). For both MSC and EPC exposed to a concentration of
30 nM DHT for at least 90 hours the proliferation and survival rate increased significantly (20 %)
above the values obtained for non-treated cells. The functional test for the capacity of cells to form
capillary tube, the Matrigel assay, revealed that the DHT- stimulated EPCs were able to form capillary
tubes in a shorter time (3h) than the control cells (6 h). Treatment with DHT on both cell types
influenced the pro-angiogenic gene expression, such as Angiogenin, CAV-1, Endostatin, eNOS, PlGF
and VEGF-A.
Conclusions DHT affects positively the morphological and functional development of EPC and
MSC, increase their proliferation and survival time and have a pro-angiogenic effect. The data
obtained can be used further on angiogenic studies and may constitute a promising alternative
therapeutic strategy.
Keywords Human mesenchymal stem cells, endothelial progenitor cells, dihydrotestosterone
angiogenesis profile.
Acknowledgements This work was supported by the Swiss–Romanian Cooperation Programme
SNSF grant no. IZERO_142213/1 and RSRP no 21 (CNCS-UEFISCDI) and by the Romanian
Academy.
A.37 - A TaqMan-based method for Haptoglobin genotyping
W Renner, R Jahrbacher, EA Marx-Neuhold, B Zulus.
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, 8036
Graz, Austria.
Background: Haptoglobin is an acute phase plasma protein and synthesized primarily in the liver and
secreted into the bloodstream. The molecule is a potent antioxidant with the main function of
scavenging free hemoglobin, which is toxic to cells. Furthermore , direct angiogenic, antiinflammatory, and immunomodulatory properties of haptoglobin have also been reported, suggesting a
potential role in malignant as well as cardiovascular disease. Haptoglobin consists of two chains, the
alpha-chain and the beta-chain, deriving from a single polypeptide after proteolytic cleavage. The
alpha-chain exists in two major forms, the alpha-1-chain and the longer alpha-2-chain, which contains
a direct repeat of 63 amino acids. This structural heterogeneity is the result of an intragenic duplication
of about 1.7 kb within the Haptoglobinp gene (HP). This HP gene polymorphism leads to three
principal genotypes, HP 1-1, 1-2, and 2-2, which have biologically important differences in their
antioxidant, scavenging, and immunomodulatory properties. Previous Haptoglobin genotyping
methods based on conventional PCR required the amplification oft he 1.7 kb duplicated region and
time-consuming laborious post-PCR processing steps.
Objective: Development and validation of a TaqMan-based method for genotyping oft he haptoglobin
1-2 polymorphism.
Design: The duplicated region oft he HP 2 allele differs from the HP 1 allele by a few single
nucleotides. We designed TaqMan probes adressing these nuclotides to detect the presence of one (HP
1-2 genotype) or two (HP 2-2 genotype) alleles with duplicated regions. The absence of the nucleotide
specific fort he duplication indicated the presence of a HP 1-1 genotype. To avoid co-amplification of
the highly homologous gene for the haptoglobin-related-protein (HRP), HP-specific primers were
selected.
Results: Genotyping of DNA samples resulted in distinct clusters indicating specific HP genotypes.
Genotype data were confirmed by sequencing as well as restriction-fragment analysis.
Conclusions: Genotyping oft he Haptoglobin 1-2 polymorphism using TaqMan-probes is fast,
economic and reliable.
A.38 - Interactome–regulome–transcriptome integrative approach as a mean to disclose cancer
stem cells regulatory circuits
C Rioualen1,2,3,4, R El-Helou1,2,3,4, E Charafe-Jauffret1,2,3,4, C Ginestier1,2,3,4, G Bidaut1,2,3,4
1
2
Centre de Recherche en Cancérologie de Marseille, Inserm U1068,
CNRS UMR7258,
3
4
Aix-Marseille Université,
Institut Paoli-Calmettes, Marseille, 13009, France.
Background: Several attempts have been made to build signatures for the prognosis of breast cancer
(BC). However, these signatures showed a low reproducibility. Thus the idea of using high-throughput
data integration rose, and more stable signatures were obtained1.
Objective: We decided to use this approach in order to identify bi-modal genes regulated by miR-600
in breast cancer stem cells (CSC).
Design: After a functional whole genome screen of an miRNA library, miR-600 was identified as a
modulator of CSC self-renewal and differentiation. We measured gene expression in CSCs after miR600 overexpression and knock-down.
We then applied a network-based integrative approach using a heavily modified version of the ITI
algorithm1. ITI has been initially developed in order to perform gene-expression-based tumor
classification, using cancer cells expression and a map of publicly available protein-protein
interactions data.
In order to isolate pathways involved in CSC evolution, we added a regulation map (regulome made of
TF-target couples), using interactions from Tred and Transfac.
In this new algorithm, the interactome and the regulome are separately investigated, using target genes
as “seeds”. If the seed's score is above a given threshold it is put aside. Its neighboring nodes are
aggregated recursively if they improve the subnetwork's score, calculated by averaging screening
scores of genes it contains. It is then statistically validated by comparing its score to those of
subnetworks generated with randomized data. Interactome and regulome subnetworks are then merged
by overlap. Resulting “metasubnetworks” are believed to be regulation pathways involved in CSC
survival or death.
Results: This two-step integration revealed interesting modules, including genes known to regulate
Wnt signaling pathways. A Gene Ontology enrichment search further suggested that miR-600 could be
a switch in the regulation of CSCs.
Conclusions: Integrating gene expression data and network information allows the identification of
biological pathways involved in CSC biology. These findings constitute a basis for anti-CSC drug
target discovery in BC.
Acknowledgements: Support to this project is coming from the Institut National du Cancer grant
Number INCA_5911 to CG.
Reference: Garcia M, Millat-Carus R, Bertucci F, Finetti P, Birnbaum D, Bidaut G. Interactometranscriptome integration for predicting distant metastasis in breast cancer. Bioinformatics. 2012 Mar
1;28(5):672-8.
A.39 - Vascular Endothelial Growth Factor linking angiogenesis, inflammation and
cardiovascular diseases.
A Saleh1,*, MG Stathopoulou1,*, M Azimi-Nezhad1, 2, NC Ndiaye1, H Murray3, C Masson1, J Lamont3,
P Fitzgerald3, S Visvikis-Siest1
UMR INSERM U 1122, IGE-PCV ‘‘Interactions Gène-Environnement en Physiopathologie Cardio
Vasculaire’’, Université de Lorraine, Nancy, F-54000, France, 2Department of Medical Genetics,
School of Medicine, Mashhad University of Medical Sciences, Mashhad, 91375-3316, Iran, 3Randox
Laboratories, Crumlin, BT29 4QY, UK.
1
Background: Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis in many in
many physiological and pathological processes, including vascular pathologies, cardiovascular
diseases (CVDs), several types of cancer, and immune-inflammatory diseases. More than 14 isoforms
grouped into two main families are expressed from the VEGF gene by differential splicing of premRNA, the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family. We have
developed an integrative approach for the study of VEGF that combines the identification of genetic
variants associated with VEGF and their relationship with intermediate phenotypes of CVD in
genetics and transcriptomic levels.
Objective: The aim was to investigate the complex relationships between VEGF and CVDs risk
factors (blood lipids, metabolic syndrome, adhesion and inflammation molecules, and blood pressure)
in the healthy.
Design: Four polymorphisms explain up to 50% of VEGF inter-individual variability1. We have
investigated their associations with ICAM-1, VCAM-1, E-, L-, P-selectins, TNF-a, CRP and IL-6
levels and blood lipids, as well as their gene × environment and epistatic interactions in 403 healthy
unrelated adults. The relationships between VEGF and these molecules were assessed in plasma and
gene expression levels.
Results: VEGF was associated with ICAM-1 and E-selectin in plasma. VEGF145 mRNA was
associated with ICAM-1, L-selectin and TNF-a expression. Interactions of the genetic variants were
shown to affect ICAM-1, E-selectin, IL-6 and TNF-a levels, while rs4416670 was associated with Lselectin expression. The polymorphism rs6921438 was associated with HDL-C (P= 1.2 × 10-7) and
LDL-C (P overall = 1.5 × 104). We also identified a significant association between the interaction
rs4416670×hypertension for ApoE levels (P= 1.7 × 105).
Conclusions: These findings support the existence of complex relationships between angiogenic,
adhesion and inflammation molecules that exist even in non-pathological situations. They may
promote the understanding of the molecular mechanisms and processes that mediate complex
pathologies such as CVDs and cancer.
Acknowledgements: This work was funded through the Collaborative BioIntelligence Program.
Reference: 1. Debette* S, Visvikis-Siest S*, Chen MH, Ndiaye NC, Song C, Destefano A, Safa R,
Azimi Nezhad M, Sawyer D, Marteau JB, Xanthakis V, Siest G, Sullivan L, Pfister M, Smith H, Choi
SH, Lamont J, Lind L, Yang Q, Fitzgerald P, Ingelsson E, Vasan RS, Seshadri S. Identification of cisand trans-acting genetic variants explaining up to half the variation in circulating vascular endothelial
growth factor levels. Circ Res 2011;109:554-63.
* equal first authors.
A.40 - Genetic determination of leukocyte telomere length in children differs from adults.
MG Stathopoulou1, AM Petrelis1, JL Buxton2, P Froguel3,4, AIF Blakemore2, S Visvikis-Siest1,5
1
UMR INSERM U1122; Interactions Gène-Environnement en Physiopathologie Cardio-Vasculaire
(IGE-PCV), Université de Lorraine, Nancy, F-54000, France,
2
Section of Investigative Medicine, Department of Medicine, Imperial College London, London W12
0NN, UK,
3
UMR CNRS 8199, Lille Pasteur Institute, Lille, F-59000, France,
4
Genomic Medicine, Imperial College London, Hammersmith Hospital, London, W12 0NN, UK,
5
Geriatric Service, Nancy University Hospital, Nancy, F-54511, France
Background: Telomeres are responsible for the protection of genomic integrity. The loss of several
base pairs from telomeres during each mitotic division leads to telomere shortening which after a
critical limit results in telomere dysfunction that triggers cell cycle arrest, apoptosis and/or cellular
senescence. Leukocyte telomere length (LTL) has been linked with a variety of human diseases,
ranging from rare monogenic disorders to age-related complex disease such as cancer, cardiovascular
diseases (CVDs) and CVD risk factors such as obesity, hypertension and diabetes 1. LTL is a highly
heritable trait, as indicated by previous studies (40-80%). Genome-wide association studies (GWAS)
have identified genetic variants which are associated with LTL, however, these studies are limited to
adult populations. Nevertheless, childhood is an extremely crucial period for the determination of LTL
and the assessment of age-specific genetic determinants, although neglected, could be of great
importance.
Objective: The objective was to provide insights and preliminary results on the neglected field of
genetic determinants of LTL in children compared to adults.
Design: In a population of healthy children (n=322, age range=6.75 - 17 years) with available GWAS
data (Illumina Human CNV370-Duo array), the LTL was measured using multiplex quantitative realtime PCR. Linear regression models adjusted for age, gender, parental age at child’s birth and body
mass index were used to test the associations of LTL with polymorphisms identified in adult GWAS
and to perform a discovery-only GWAS.
Results: Among the 21 polymorphisms previously shown to be associated with adults LTL through
GWAS methodologies none was associated with LTL in our paediatric sample. Furthermore, our
GWAS approach demonstrated 6 novel variants that reached the level of suggestive association (P ≤ 5
×10-5) and explain a high percentage of children’s LTL.
Conclusions: It appears that the study of genetic determinants of LTL in children may identify novel
variants not previously identified in adults. Studies in large-scale children populations are needed for
the confirmation of these results, possibly through a childhood consortium that could better handle the
methodological challenges of LTL genetic epidemiology field.
Acknowledgements: This work was funded through the Collaborative BioIntelligence Program.
Reference: 1. Calado RT, Young NS. Telomere diseases. N Engl J Med 2009;361:2353-65
A.41 - Effect of carotenoids caspanthin and β-cryptoxanthin on B16 murine melanoma cells
T. Tomic1,4, M. O. Villareal2, J. Han2,3, D. Margout4 , M. Larroque4, H. Isoda2,3
1
Graduate School of Life and Environmental Sciences, University of Tsukuba,
Alliance for Research on North Africa (ARENA), University of Tsukuba,
3
Faculty of Life and Environmental Sciences, University of Tsukuba,Tennodai 1-1-1, Tsukuba City,
305-8587 Japan;
4
Faculty of Pharmacy, University of Montpellier I, 5 bd Henri IV - CS 19044, 34967 Montpellier
Cedex 2, Montpellier, France
2
Background: Metastatic melanoma is the most invasive and deadly form of skin cancer with no
effective therapy to treat advanced disease. It is one of the most aggressive and highly proliferative
human malignancies with a median survival of only 6–9 months once distant sites become seeded
from skin. The important obstacle in finding efficient therapeutics has been the relatively poor
understanding of biochemical pathways that govern proliferation and survival of melanoma cells.
In addition, current anti-melanoma drugs either do not reach their targets or have to be administrated at
dosing regimens that result in unbearable toxicities to normal cellular compartments. In addition to
active prevention and early detection of melanomas, it appears necessary to develop a new effective
and safer systemic therapy for melanoma patients.
Epidemiological investigations have shown that cancer risk is inversely related to the consumption of
green and yellow vegetables and fruits. Thus, we have carried out more extensive study on potential
antimelanoma effect of carotenoids, the most abundant groups of natural pigments, found in numerous
fruits and vegetables with strong antioxidant activity.
Objective: In this study, we investigated the effect of pigments found in paprika, capsanthin and cryptoxanthin, on B16 murine melanoma cell viability.
Design: To determine whether these compounds affect B16 cell viability, we analyzed the percentage
of cell death, by MTT assay and their effects on the cell cycle were evaluated. Furthermore, to
determine their regulatory effect on intracellular signaling mediated by MAPKs, western blotting for
ERK, JNK and p38 was done. Determination of the expression of cancer-related genes was also
undertaken.
Results and Conclusion: In our study, for the first time we demonstrated that capsanthin and cryptoxanthin inhibits melanoma cell growth by interfering with MAPKs. The results of this study
suggest that ingestion of paprika and other vegetables rich in capsanthin and - cryptoxanthin might
have impart a protective effect against skin cancer.
Keywords: capsanthin, -cryptoxanthin, melanoma, B16 cells, MTT assay
A.42 - Drug exposure dependent gene expression changes in the chemoimmune system
including transporters, metabolic enzymes, and nuclear receptors
H Tordai1,2, K Jakab1,2, D Szöllősi1,2, B Sarkadi1,2,3, T Hegedűs1,2
1
MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Budapest, 1094,
Hungary; 2Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, 1094,
Hungary; 3Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, 1117,
Hungary
Background: Multidrug ABC transporters have been indicated as major factors in cancer multidrug
resistance (MDR). While several efficient MDR transporter inhibitors have been developed, failure of
clinical trials indicated that modulating the function of the transporters is insufficient to eliminate
cancer drug resistance. Transporters are part of the chemoimmune/chemodefense system, which
neutralizes toxic and therapeutic compounds entering our body. This system also includes metabolic
enzymes and nuclear receptors, which sense xenobiotics activating the transcription of enzymes and
transporters. The modulation of the chemoimmune system is hindered by the lack of knowledge on the
function of multidrug binding transporters, enzymes and receptors as a coherent network.
Objective: To learn the network level function of the chemoimmune system we study the cooperation
and co-regulation of transporters, metabolic enzymes and nuclear receptors by analyzing mRNA
expression changes upon drug treatment in various cell lines.
Design: We constructed a pipeline to analyze the results of mRNA chip experiments of human cell
lines treated with over 1,300 different chemical compounds, publicly available in the gene expression
database of Broad Institute. Raw microarray data were background corrected, normalized and
expression changes at different thresholds were calculated. mRNA expression changes of drug
transporters, metabolizing enzymes, and nuclear receptor genes were extracted and experiments were
clustered based on the expression patterns of these chemoimmune genes. The grouped expression
patterns were correlated with data on chemical structure, metabolism and transport of the drugs used in
the experiments.
Results: Similar drugs caused similar chemoimmune gene expression patterns in different cell lines
suggesting that a given type of drug causes specific changes in the gene expression patterns.
Preliminary pathway analysis indicates that in many times drugs induce the expression of proteins
taking part in GSH-metabolism (e.g. cysteine importer SLC7A11, glutamate cysteine ligase for GSH
synthesis) which play an important role in detoxification.
Conclusion: Our results can be applied to infer metabolic and regulatory pathways in the
chemoimmune system.
Acknowledgements: The support of KTIA-AIK-12-2012-0025 is gratefully acknowledged.
A.43 - Extensive pathogenicity of mitochondrial heteroplasmy in healthy human individuals
K. Yea, J. Lua,b, F. Mac, A. Keinand, Z. Gua
a
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, USA
State Key Laboratory of Protein and Plant Gene Research, Center for Bioinformatics, College of Life
Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
c
Department of Medical Oncology, Cancer Hospital, Chinese Academy of Medical Sciences, Beijing
100021, China
d
Department of Biostatistics and Computational Biology, Cornell University, Ithaca, New York 14853,
USA
b
A majority of mitochondrial DNA (mtDNA) mutations reported to be implicated in diseases are
heteroplasmic, a status with co-existing mtDNA variants in a single cell. Quantifying the prevalence of
mitochondrial heteroplasmy and its pathogenic effect in healthy individuals could further our
understanding of its possible roles in various diseases. 1085 human individuals from 14 global
populations have been sequenced by the 1000 Genomes Project to a mean coverage of ~2000X on
mtDNA. Using a combination of stringent thresholds and a maximum likelihood method to define
heteroplasmy, we demonstrated that ~90% of the individuals carry at least one heteroplasmy. At least
20% individuals harbor heteroplasmies reported to be implicated in disease. Mitochondrial
heteroplasmy tend to show high pathogenicity, and is significantly over-represented in diseaseassociated loci. Consistent with their deleterious effect, heteroplasmies with derived allele frequency
larger than 60% within an individual show a significant reduction in pathogenicity, indicating the
action of purifying selection. Purifying selection on heteroplasmies can also be inferred from nonsynonymous and synonymous heteroplasmy comparison and the unfolded site frequency spectra for
different functional sites in mtDNA. Nevertheless, in comparison to population polymorphic mtDNA
mutations, the purifying selection is much less efficient in removing heteroplasmic mutations. The
prevalence of mitochondrial heteroplasmy with high pathogenic potential in healthy individuals, along
with the possibility of these mutations drifting to high frequency inside a subpopulation of cells across
life-span, emphasizes the importance of managing mitochondrial heteroplasmy to prevent disease
progression.
A.44 - Association of col5a1, mmp3, col1a1 genes with injuries during sports activity in a greek
population of 739 subjects.
Zouli K., Drakoulis N.
National and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy.
Background and objectives: Tendon and ligaments within the upper and lower limbs are some of the
more common sites of musculoskeletal injuries during physical activity. Several extrinsic and intrinsic
factors have been shown to be associated with these injuries. More recently, studies have suggested
that there is also, at least in part, a genetic component to the Achilles tendon (AT) and anterior cruciate
ligament (ACL) injuries. Sequence variants of the COL5A1, MMP3 and COL1A1 genes showed
association with Achilles tendinopathies and ACL ruptures. Those genes encode for proteins directly
involved in biological processes within tendons and ligaments.
Methods: In this genetic association study, the 739 recruited Greek participants consisted of 378
females (51,2%) and 361 males (48,8%) aged from 1 to 94 years. The participants were genotyped for
the COL5A1 rs12722 (C/T), MMP3 rs679620 (A/G) and COL1A1 rs1800012 (G/T) sequence
variants. The cohort was divided into 7 subgroups according to the SNP or/and the SNPs combination
examined.
Results: The vast majority of genotype combinations appeared to have a different impact on the risk
of each injury. The genotype combination CT+AG+GG, (over-represented among our population
within the group VII, 14,9%), was associated with increased risk of ACL rupture (GS=4), although it
tended to have a protective effect against Achilles tendinopathy (GS=1). Furthermore, within the same
group, the protective CC+AG+TT1A1 combination and the hazardous TT5A1+GG+GG1A1 combination
were underrepresented (0,3% and 5,8%, respectively). Similar findings were identified within the
other 6 subgroups.
Conclusions: This study demonstrated that chronic Achilles tendinopathy and ACL ruptures are
distinctly different injuries with genetic predisposition. Only a minority of the Greek population
examined seemed to be protected for both injuries. Finally, individuals at increased risk of both ATP
and ACL ruptures were underrepresented among the cohort.
References: Zouli K., N. Drakoulis. Association of COL5A1, MMP3, COL1A1 genes with injuries
during sports activity in a Greek population. In: N. Drakoulis (ed.) Contribution of Genetic Analyses
in Predisposition, Diagnosis, Prognosis and Therapeutic Approach to Disease, Chapter 5, I. Levakos,
N. Papaioannou (eds.) Orthopedics, sports medicine. APR-Publicartions, Athens 2013, pp 216-223.
A.45 - Identifying the clinical, environmental and genetic predictive factors for epilepsy
management: DICE Study
Antonio Gil-Nagel1, Mar Carreño2, Carlos Cara3, Juan Jose Poza4, Juan Carlos Sánchez5, Juan Ramón
González 6, Pedro Serrano 7.
1
Hospital Ruber Internacional, Madrid; 2Hospital Clínic i Provincial de Barcelona, Barcelona; 3UCB
Pharma, Madrid; 4Hospital de Donostia, San Sebastián; 5Hospital Clínico San Cecilio, Granada;6
Centro de Investigación en Epidemiología Ambiental (CREAL), Barcelona; 7 Hospital Torre
Cárdenas, Almería.
Purpose: To determine clinical, environmental and genetic factors related to DICE.
Method: Cross-sectional, epidemiological, case-control, multicenter study to analyse predictive
factors related to DICE in patients (≥18 years) with focal epilepsy. Demographic, environmental and
clinical data, plus DNA samples were collected. Univariate analysis identified factors that were most
important/predictive characteristics and included a binary logistic regression model. Goodness-of-fit
was evaluated using Hosmer-Lemeshow statistics; accuracy assessed via bootstrapped area under the
Receiver Operating Characteristic (ROC) curve (AUC) with 95% CI.
Results: Among 564 patients (mean age 42.9 years; 52.3% women), 48.6% had controlled epilepsy.
Using a multivariate stepwise procedure 5 SNPs (CYP3A4, GSTM3, GSTP1, DRD3 and GSTT1) were
selected to create an epilepsy-risk allele score based on the sum of risk alleles. Lower frequency
variant alleles of each polymorphism were associated with controlled epilepsy. On average, each risk
allele conferred an estimated increased risk of controlled epilepsy equal to 32% (OR per allele = 1.32;
CI95% = 1.13, 1.55, p=4.0x10-4). The clinical variables that best predict the probability of controlled
epilepsy were: focal seizures with secondary generalization (CRFO), OR=2.32 (1.49, 3.64; p=2.1x104
): age at diagnosis, 1.04 (1.03, 1.06; p=3.1x10-08). These genes in a constructed genetic score had
predictive power of ~60% (AUC=0.599); cross-validation demonstrated AUC (bootstrap] of 0.56.
Including the clinical variables increased predictive power to 75% (AUC=0.755)
Conclusions: The CYP3A4, GSTM3, GSTP1, GSTT1 and DRD3 allele variations (plus
focal seizures with secondary generalization, and age at diagnosis)
were most predictive of controlled epilepsy (CE)..
Acknowledgement: Funded by UCB Pharma
A.46 - Antihypertensive and Vasodilatory Tripeptides from Milk Casein; Possible Role of
Bradykinin.
J. Nussberger1, A. Dubach2, T. Mattsson3, R. Korpela4, S. Järvenpää4, A. Siltari4, H. Vapaatalo4
University Hospital Lausanne1, Emmi Schweiz AG,2 Emmen, Switzerland, Valio Ltd3, Helsinki,
University of Helsinki4, Finland
Background: Biologically active tripeptides (Ile-Pro-Pro and Val-Pro-Pro) in fermented milk products
lower blood pressure (BP) and reduce arterial stiffness in animal and human studies. These effects are
related to inhibition of angiotensin converting enzyme (ACE-1) and thus reduced production of potent
vasoconstrictive angiotensin II (AII).
Objective: The role of increased vasodilatory bradykinin (BK) seen in animals studies is not known
in
humans.
The
present
study
was
aimed
to
answer
this
question.
Design: Twelve normotensive, non-smoking healthy volunteers participated in this double-blind,
randomized cross-over trial. On the first day (D 1) the subjects took either placebo or fermented test
drink containing 5 or 50 mg (the sum amount) of the two peptides. Physiological variables (e.g. blood
pressure, heart rate) were followed for 9 h, and urine collected for clinical chemistry. The procedure
was repeated after 28 days consumption of the drinks in randomized order. BK levels in plasma were
the main outcome (Nussberger, J. & al.. Lancet 1998: 351:1693-7). Other variables related to renin -
angiotensin - aldosterone system (RAAS), vasodilatory prostaglandins and nitric oxide were analyzed
using commercial kits.
Results: Blood pressure or heart rate of the normotensive subjects behaved similarly in all acute
treatment sessions (D 1 or D 28). The main outcome, plasma BK concentrations showed marked interand intraindividual variation . No statistically significant effect of the peptide drink vs placebo was
seen in the 28 day intervention. However the acute effect showed up to three fold higher
concentrations vs the basal level in 5 h samples both on D 1 (1.55 to 4.76 fmol/l) and D 28 (1.49 to
4.05 fmol/l). When the acute effect of the peptide drinks was presented as AUC minus baseline, a clear
positive linearity was seen between the doses on D 1. Plasma renin activity, trapping ACE activity,
aldosterone or urine excretion of indicators of prostaglandin or nitric oxide systems showed no
significant
treatment
effects.
Conclusions: In normotensive subjects, the use of drinks containing ACE inhibitory peptides, Ile-ProPro and Val-Pro-Pro
neither in acute nor in prolonged intervention showed significant
antihypertensive effect or changes in RAAS, prostaglandin or nitric oxide systems, although ACEinhibition was indicated as increased plasma bradykinin levels.
A.47 - Evidence for integrating point of care diagnostics as an integral part of primary
healthcare in resource-limited settings in South Africa: a people-centred health systemstrengthening approach
T.P. Mashamba-Thompson, N. Moodley, M.M. Moshabela
Discipline of Rural Health, School of Nursing and Public Health, University of KwaZulu Natal
Background: It has been estimated that the deployment of new point of care (POC) diagnostics could
prevent more than 1.2 million deaths in the developing world.
Objective: We conducted a scoping review on the impact of POCT compared to laboratory tests for
enabling the following patient centred outcomes in resource-limited settings.
Methods: A detailed search on NCBI, WED of Science and EBSCOhost for relevant publications
from inception to current, combined into phrases including Boolean (AND, OR) using a keyword
searches with the terms “rural communities”,“ point of care testing”, “ early diagnosis ” , “ improved
healthcare outcome” and “access to healthcare”.
Results: 14 studies met the inclusion and quality criteria. There appears to be growing interest in the
integration of POCT for management of HIV, detection of HIV co-infections. However, evidence to
date, does not adequately address other components that are crucial to the sustainability of POCT
services such as: accessibility; availability; acceptability; reliability of POC diagnostics in resource
limited settings as well knowledge and skill level of current POCT amongst all stakeholders.
Conclusion: Further evaluation of current POC diagnostics is necessary prior to adoption and scale up
of POCT in South Africa’s resource-limited settings.
Reference:
Nitika Pant Pai et al(2012). "Point-of-Care Testing for Infectious Diseases: Diversity, Complexity, and
Barriers in Low- And Middle-Income Countries " PLOS Medicine 9.