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RNA-interference based screen identifies new factors important for NF-kappaB activation and termination
New Biotechnology · Volume 27S · April 2010
POSTERS ABSTRACTS 2
[P2.30] Unique spectrum of spast variants in Estonian HSP patients: presence of benign missense changes but lack of exonic rearrangements R. Tamm 1,3,4,∗ , M. Ferrero 1,3,4
Braschinsky 2,3,4 , C.
Beetz 2,3,4 , E.
Sachez-
1
University of Tartu, Estonia University Hospital Jena, Germany 3 Hospital Central Universitario de Asturias, Spain 4 Estonian Biocentre, Estonia 2
Hereditary spastic paraplegia is a clinically and genetically heterogeneous disorder that may be inherited in an autosomaldominant, autosomal-recessive or X-linked fashion. The most common autosomal-dominant form of the disease is due to the mutations in the SPAST gene. The aim of this study was to detect new sequence variants within the SPAST gene and to describe the phenotypes of the patients with hereditary spastic paraplegia in Estonia that corresponds to SPAST gene mutations. Forty-nine patients and 50 healthy control individuals with no family history of the disease participated in this study. Initially, all samples were screened by denaturing high performance liquid chromatography (DHPLC), for rearrangement detection multiplex ligation-dependent probe amplification (MLPA) assay was used and all samples with abnormal DHPLC and MLPA profiles were sequenced. Controls were sequenced for the same gene regions as patients. Nineteen (38.8%) out of 49 patients had sequence variant in SPAST gene. There was one sporadic case. All patients had pure hereditary spastic paraplegia. Twelve changes in the SPAST gene (c.131C > T, c.484G > A, c.685A > G, c.1174-1G > C, c.1185delA, c.1276C > T, c.1245 + 202delG, c.1245 + 215G > C, c.1352-1356delGAGAA, c.1378C > A, c.1518 1519insTC, c.1841 1842insA) were detected, two of which was described previously (c.131C > T, c.1245 + 202delG). Three mutations (c.131C > T, c.484G > A c.685A > G) did not show familial segregation and were considered as benign missense variants. This study expands the spectrum of variations of the SPAST gene, including benign missense variants and short INDELs. In contrast to other studies, no large rearrangements were found. We suggest seven new probably pathogenic variants in our study group. doi:10.1016/j.nbt.2010.01.193
[P2.31] Functional genomics and molecular docking for effective drug discovery against H1N1 virus A. Sharma ∗ , A. Tendulkar, K.R. Reddy, P. Wangikar Indian Institute of Technology, India
Genome of H1N1 virus has been sequenced (http:// www.ncbi.nlm.nih.gov/genomes/FLU/SwineFlu.html) and the structure of the viral neuraminidase was obtained by homolS68
www.elsevier.com/locate/nbt
ogy modelling [1]. The neuraminidase is responsible for virus attachment to the host cell. We analyzed the modelled structure using the state of the art functional genomics and molecular docking to propose a couple of effective drugs. We initially obtained putative functional sites of the enzyme using prediction servers such as PINTS and PROFUNC. Several antiviral compounds were docked against the enzyme around the putative functional site using PatchDock and GemDock. The compounds were selected from chemical databases (e.g. ZINC, PUBCHEM, DRUG BANK and CHEMDB) and from antiviral plant metabolites of several medicinal plants. The experiment reveals that the plant metabolites such as Hesperidin (score: 6080) and narirutin (score: 5628) from medicinal plant Citrus junos v. Tanaka, Rutaceae [2] produced the highest docking score as compared to traditional drugs like Oseltamivir (Tamiflu) (score: 4240). Gemdock also produces top rank for Hesperidin (−121.615)and Narirutin (−114.740) as compared to Oseltamivir (−79.738). These are known flavonoid compounds and mainly inhibit the activity of neuraminidase enzyme and the membrane fusion in Influenza A virus. Thus, our findings establish that Hesperidin and Narirutin can be more potent antiviral drugs against H1N1 virus as compared to Oseltamivir. References 1 Maurer-Stroh, S. et al. (2009) Mapping the sequence mutations of the 2009 H1N1 influenza A virus neuraminidase relative to drug and antibody binding sites. Biol. Direct 4, 1–9 2 Wang, X. et al. (2006) Anti-influenza agents from plants and traditional Chinese medicine. Phytother. Res. 20, 335–341 doi:10.1016/j.nbt.2010.01.194
[P2.32] RNA-interference based screen identifies new factors important for NF-kappaB activation and termination S. Bartfeld ∗ , C. Rechner, B. Bauer, S. Hess, A. Maeurer, N. Machuy Max Planck Institute for Infection Biology, Germany
Research efforts over the past decades have led to a more and more detailed understanding of NF-kappaB signaling but the picture is not yet complete. Especially the activation of NF-kappaB by bacteria and the termination of NF-kappaB activation are not well understood. To identify new key regulators of NF-kappaB we have conducted an RNA-interference based screen. For this purpose, we have developed a new model for high throughput analysis using a human epithelial cell line stably expressing a p65–GFP-fusion construct. The nuclear translocation of p65–GFP can be quantified by automated microscopic analysis. Three different stimuli were compared: the cytokines TNFalpha and IL-1beta and the gastric pathogen Helicobacter pylori. Screening a library of siRNAs targeting 646 kinases and associated proteins, we identified 24 factors, which also included known factors such as IKKalpha and IKKbeta. Interestingly, we found general NF-kappaB regulators as well as factors specifically acting in one pathway. Amongst the newly discovered regulators, an ubiquitin E3 ligase could be shown to be necessary
New Biotechnology · Volume 27S · April 2010
for the termination of NF-kappaB activation. Upon knockdown of this E3, base level of IkappaBalpha is reduced and nuclear translocation of p65 as well as IKK activity is prolonged. The screen has been expanded to a genome-wide scale. doi:10.1016/j.nbt.2010.01.195
[P2.33] Towards a genetic screening test for dyslexia allowing functional regeneration: a strategy for identification and analysis of genetic risk factors K. Holger 1,2,∗ , A. Wilke 1,2 , P. Ahnert 3 , J. Boltze 1
POSTERS ABSTRACTS 2
lower for conserved elements associated with imprinted regions. Additionally, these elements are substantially enriched in overlap with conserved repetitive elements, prominently LINE-1 repeats. Most interestingly, we observed a low conservation of conserved elements in coding exons of genes with a maternal expression pattern. The enrichment of intronic CpG islands supports their role as epigenetic key regulatory elements [3]. We suggest that evolutionary conserved LINE-1 elements fulfill regulatory functions in imprinted regions as well, similar to the situation on the X chromosome. Reduced CpG deamination rates may indicate a hypomethylation of imprinted regions relative to the whole genome, whereas the low conservation of maternally expressed genes hints at a specific mode of evolution.
1
TRM Leipzig, Germany IZI Fraunhofer Leipzig, Germany 3 IMISE, Germany 2
Our aim is to develop a genetic screening test for dyslexia, a severe disorder of reading and frequently of writing, affecting approx. 4% of all schoolchildren. A significant problem is late diagnosis resulting in a decreased chance of functional regeneration. Our solution is an early genetic test that will initiate existing early training programs. Genetic dyslexia markers necessary for this test are identified in a microarray based fine screen supplemented by polymorphisms of highly relevant candidate genes. Validation of these markers is done by (A) genotyping an independent cohort; (B) by characterising markers in functional magnet resonance imaging (fMRI) and electroencephalography (EEG); and (C) by characterising markers in allele specific mRNA-expression analysis or allele-specific chromatin immunoprecipitation (ChIP). The final test will neither include fMRI/EEG nor expression analysis. It translates genetic findings into a clinical assay. This test would allow early identification of children at risk, enabling early support resulting in functional regeneration. doi:10.1016/j.nbt.2010.01.196
[P2.35] Conserved elements in imprinted genes B. Hutter ∗ , M. Bieg, V. Helms, M. Paulsen Saarland University, Germany
Imprinted genes are monoallelically expressed with the choice of the active allele depending on its parental origin. Owing to their association with differentially methylated regions, they serve as a model for epigenetic regulation. To decipher the particular DNA sequence features that distinguish imprinted genes, we compared evolutionarily conserved elements [1] associated with 58 imprinted genes to those of all genes in the human genome. Conserved elements in imprinted genes are enriched in CpGrich motifs. They also overlap more frequently with CpG islands, especially intronic ones, than those in biallelically expressed genes. Investigating the (TpG + CpA)/(2*CpG) ratio, which serves as an estimate for CpG deamination effects [2], we found it to be
References 1 Siepel, A. et al. (2005) Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 2 Hutter, B. et al. (2009) Identifying CpG islands by different computational techniques. Omics 13, 153–164 3 Hutter, B. et al. (2006) Tandem repeats in the CpG islands of imprinted genes. Genomics 88, 323–332 doi:10.1016/j.nbt.2010.01.197
[P2.36] Differential allelic expression of genes associated with asthma J. Burkhardt 1,∗ , H. Kirsten 2,3 , G. Wolfram 3 , E. Quente 2,3 , P. Ahnert 4 1
University Leipzig, Germany Fraunhofer IZI Leipzig, Germany 3 TRM Leipzig, Germany 4 IMISE Leipzig, Germany 2
An important question in ongoing research of genetic traits in complex diseases is functional relevance of identified disease associating genetic factors. Differential allelic expression (DAE) has been estimated to affect 20–50% of human genes and can be measured by genotyping cSNPs in heterozygous cDNA samples. Coding SNPs in IL13 and CSF2 were associated with asthma. We investigated whether those cSNPs are correlated with cis-directed expression of a specific allele. Results may provide a functional link between specific genetic variants and specific pathological mechanism related to asthma. One coding SNP per gene was investigated. About 50 immortalized B cell-lines were screened for heterozygosity. Genomic DNA (gDNA), RNA and cDNA were won. To identify DAE, a mass-based genotyping system was used for quantitative genotyping of both alleles. Significant derivation of allelic ratios between cDNA and gDNA samples indicated allelic regulation. Similar direction of intra-sample effects for a given SNP indicated cis-regulation. Both SNPs showed significant DAE (P[IL13] = 0.002, P[CSF2] = 0.01). www.elsevier.com/locate/nbt S69
POSTERS ABSTRACTS 2
[P2.30] Unique spectrum of spast variants in Estonian HSP patients: presence of benign missense changes but lack of exonic rearrangements R. Tamm 1,3,4,∗ , M. Ferrero 1,3,4
Braschinsky 2,3,4 , C.
Beetz 2,3,4 , E.
Sachez-
1
University of Tartu, Estonia University Hospital Jena, Germany 3 Hospital Central Universitario de Asturias, Spain 4 Estonian Biocentre, Estonia 2
Hereditary spastic paraplegia is a clinically and genetically heterogeneous disorder that may be inherited in an autosomaldominant, autosomal-recessive or X-linked fashion. The most common autosomal-dominant form of the disease is due to the mutations in the SPAST gene. The aim of this study was to detect new sequence variants within the SPAST gene and to describe the phenotypes of the patients with hereditary spastic paraplegia in Estonia that corresponds to SPAST gene mutations. Forty-nine patients and 50 healthy control individuals with no family history of the disease participated in this study. Initially, all samples were screened by denaturing high performance liquid chromatography (DHPLC), for rearrangement detection multiplex ligation-dependent probe amplification (MLPA) assay was used and all samples with abnormal DHPLC and MLPA profiles were sequenced. Controls were sequenced for the same gene regions as patients. Nineteen (38.8%) out of 49 patients had sequence variant in SPAST gene. There was one sporadic case. All patients had pure hereditary spastic paraplegia. Twelve changes in the SPAST gene (c.131C > T, c.484G > A, c.685A > G, c.1174-1G > C, c.1185delA, c.1276C > T, c.1245 + 202delG, c.1245 + 215G > C, c.1352-1356delGAGAA, c.1378C > A, c.1518 1519insTC, c.1841 1842insA) were detected, two of which was described previously (c.131C > T, c.1245 + 202delG). Three mutations (c.131C > T, c.484G > A c.685A > G) did not show familial segregation and were considered as benign missense variants. This study expands the spectrum of variations of the SPAST gene, including benign missense variants and short INDELs. In contrast to other studies, no large rearrangements were found. We suggest seven new probably pathogenic variants in our study group. doi:10.1016/j.nbt.2010.01.193
[P2.31] Functional genomics and molecular docking for effective drug discovery against H1N1 virus A. Sharma ∗ , A. Tendulkar, K.R. Reddy, P. Wangikar Indian Institute of Technology, India
Genome of H1N1 virus has been sequenced (http:// www.ncbi.nlm.nih.gov/genomes/FLU/SwineFlu.html) and the structure of the viral neuraminidase was obtained by homolS68
www.elsevier.com/locate/nbt
ogy modelling [1]. The neuraminidase is responsible for virus attachment to the host cell. We analyzed the modelled structure using the state of the art functional genomics and molecular docking to propose a couple of effective drugs. We initially obtained putative functional sites of the enzyme using prediction servers such as PINTS and PROFUNC. Several antiviral compounds were docked against the enzyme around the putative functional site using PatchDock and GemDock. The compounds were selected from chemical databases (e.g. ZINC, PUBCHEM, DRUG BANK and CHEMDB) and from antiviral plant metabolites of several medicinal plants. The experiment reveals that the plant metabolites such as Hesperidin (score: 6080) and narirutin (score: 5628) from medicinal plant Citrus junos v. Tanaka, Rutaceae [2] produced the highest docking score as compared to traditional drugs like Oseltamivir (Tamiflu) (score: 4240). Gemdock also produces top rank for Hesperidin (−121.615)and Narirutin (−114.740) as compared to Oseltamivir (−79.738). These are known flavonoid compounds and mainly inhibit the activity of neuraminidase enzyme and the membrane fusion in Influenza A virus. Thus, our findings establish that Hesperidin and Narirutin can be more potent antiviral drugs against H1N1 virus as compared to Oseltamivir. References 1 Maurer-Stroh, S. et al. (2009) Mapping the sequence mutations of the 2009 H1N1 influenza A virus neuraminidase relative to drug and antibody binding sites. Biol. Direct 4, 1–9 2 Wang, X. et al. (2006) Anti-influenza agents from plants and traditional Chinese medicine. Phytother. Res. 20, 335–341 doi:10.1016/j.nbt.2010.01.194
[P2.32] RNA-interference based screen identifies new factors important for NF-kappaB activation and termination S. Bartfeld ∗ , C. Rechner, B. Bauer, S. Hess, A. Maeurer, N. Machuy Max Planck Institute for Infection Biology, Germany
Research efforts over the past decades have led to a more and more detailed understanding of NF-kappaB signaling but the picture is not yet complete. Especially the activation of NF-kappaB by bacteria and the termination of NF-kappaB activation are not well understood. To identify new key regulators of NF-kappaB we have conducted an RNA-interference based screen. For this purpose, we have developed a new model for high throughput analysis using a human epithelial cell line stably expressing a p65–GFP-fusion construct. The nuclear translocation of p65–GFP can be quantified by automated microscopic analysis. Three different stimuli were compared: the cytokines TNFalpha and IL-1beta and the gastric pathogen Helicobacter pylori. Screening a library of siRNAs targeting 646 kinases and associated proteins, we identified 24 factors, which also included known factors such as IKKalpha and IKKbeta. Interestingly, we found general NF-kappaB regulators as well as factors specifically acting in one pathway. Amongst the newly discovered regulators, an ubiquitin E3 ligase could be shown to be necessary
New Biotechnology · Volume 27S · April 2010
for the termination of NF-kappaB activation. Upon knockdown of this E3, base level of IkappaBalpha is reduced and nuclear translocation of p65 as well as IKK activity is prolonged. The screen has been expanded to a genome-wide scale. doi:10.1016/j.nbt.2010.01.195
[P2.33] Towards a genetic screening test for dyslexia allowing functional regeneration: a strategy for identification and analysis of genetic risk factors K. Holger 1,2,∗ , A. Wilke 1,2 , P. Ahnert 3 , J. Boltze 1
POSTERS ABSTRACTS 2
lower for conserved elements associated with imprinted regions. Additionally, these elements are substantially enriched in overlap with conserved repetitive elements, prominently LINE-1 repeats. Most interestingly, we observed a low conservation of conserved elements in coding exons of genes with a maternal expression pattern. The enrichment of intronic CpG islands supports their role as epigenetic key regulatory elements [3]. We suggest that evolutionary conserved LINE-1 elements fulfill regulatory functions in imprinted regions as well, similar to the situation on the X chromosome. Reduced CpG deamination rates may indicate a hypomethylation of imprinted regions relative to the whole genome, whereas the low conservation of maternally expressed genes hints at a specific mode of evolution.
1
TRM Leipzig, Germany IZI Fraunhofer Leipzig, Germany 3 IMISE, Germany 2
Our aim is to develop a genetic screening test for dyslexia, a severe disorder of reading and frequently of writing, affecting approx. 4% of all schoolchildren. A significant problem is late diagnosis resulting in a decreased chance of functional regeneration. Our solution is an early genetic test that will initiate existing early training programs. Genetic dyslexia markers necessary for this test are identified in a microarray based fine screen supplemented by polymorphisms of highly relevant candidate genes. Validation of these markers is done by (A) genotyping an independent cohort; (B) by characterising markers in functional magnet resonance imaging (fMRI) and electroencephalography (EEG); and (C) by characterising markers in allele specific mRNA-expression analysis or allele-specific chromatin immunoprecipitation (ChIP). The final test will neither include fMRI/EEG nor expression analysis. It translates genetic findings into a clinical assay. This test would allow early identification of children at risk, enabling early support resulting in functional regeneration. doi:10.1016/j.nbt.2010.01.196
[P2.35] Conserved elements in imprinted genes B. Hutter ∗ , M. Bieg, V. Helms, M. Paulsen Saarland University, Germany
Imprinted genes are monoallelically expressed with the choice of the active allele depending on its parental origin. Owing to their association with differentially methylated regions, they serve as a model for epigenetic regulation. To decipher the particular DNA sequence features that distinguish imprinted genes, we compared evolutionarily conserved elements [1] associated with 58 imprinted genes to those of all genes in the human genome. Conserved elements in imprinted genes are enriched in CpGrich motifs. They also overlap more frequently with CpG islands, especially intronic ones, than those in biallelically expressed genes. Investigating the (TpG + CpA)/(2*CpG) ratio, which serves as an estimate for CpG deamination effects [2], we found it to be
References 1 Siepel, A. et al. (2005) Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 2 Hutter, B. et al. (2009) Identifying CpG islands by different computational techniques. Omics 13, 153–164 3 Hutter, B. et al. (2006) Tandem repeats in the CpG islands of imprinted genes. Genomics 88, 323–332 doi:10.1016/j.nbt.2010.01.197
[P2.36] Differential allelic expression of genes associated with asthma J. Burkhardt 1,∗ , H. Kirsten 2,3 , G. Wolfram 3 , E. Quente 2,3 , P. Ahnert 4 1
University Leipzig, Germany Fraunhofer IZI Leipzig, Germany 3 TRM Leipzig, Germany 4 IMISE Leipzig, Germany 2
An important question in ongoing research of genetic traits in complex diseases is functional relevance of identified disease associating genetic factors. Differential allelic expression (DAE) has been estimated to affect 20–50% of human genes and can be measured by genotyping cSNPs in heterozygous cDNA samples. Coding SNPs in IL13 and CSF2 were associated with asthma. We investigated whether those cSNPs are correlated with cis-directed expression of a specific allele. Results may provide a functional link between specific genetic variants and specific pathological mechanism related to asthma. One coding SNP per gene was investigated. About 50 immortalized B cell-lines were screened for heterozygosity. Genomic DNA (gDNA), RNA and cDNA were won. To identify DAE, a mass-based genotyping system was used for quantitative genotyping of both alleles. Significant derivation of allelic ratios between cDNA and gDNA samples indicated allelic regulation. Similar direction of intra-sample effects for a given SNP indicated cis-regulation. Both SNPs showed significant DAE (P[IL13] = 0.002, P[CSF2] = 0.01). www.elsevier.com/locate/nbt S69