European Human Genetics Conference 2007 June 16 – 19, 2007 ...

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Genomics, technology, bioinformatics several new approaches in genetic testing are selected for further evaluation and validation in collaboration with their manufacturers and inventors.Currently we are evaluating several new techniques including High-Resolution Melting-Curve Analysis (HR-MCA) and Conformation Sensitive Capillary Electrophoresis (CSCE), two fluorescent heteroduplex-based mutation scanning-methods in collaboration with Idaho Technologies and NGRL-Wessex together with Applied Biosystems respectively and Pyrophosphorolysis Activated Polymerization (PAP), a very sensitive detection method for the presence of low mutation levels in the presence of excess wt-allele, in collaboration ServiceXS. More details on ongoing and new evaluation projects will be shown also at the EUGT-Unit 5 Satellite-meeting during this conference. P1230. Comprehensive human genetic analysis using 454 Sequencing technology B. Taillon; 454 Life Sciences Corporation, Branford, CT, United States. 454 Life Sciences has developed a revolutionary technology that can produce up to 100 megabases of sequence data on a single instrument. Many biologically meaningful and complex regions of the human genome can now be analyzed with this system without the time or cost constraints of conventional DNA sequencing methods. We will demonstrate how this technology can be applied to the study of the cancer genome through ultra-deep sequencing of genes involved in oncogenesis. The technology also allows researchers to conduct studies of human genetic variation (SNPs, indels and rearrangements) from a complex population. We will also discuss applications in the study of gene regulation through sequencing of microRNA, transcriptome and gene copy numbers. The large sequencing capacity of the 454 Life Sciences technology platform, combined with the flexibility and versatility of sample handling and data output makes it a method of choice for human molecular genetics. P1231. Characterization of a human fetal cartilage EST library focussing on transcription factors P. Hermanns 1 , C. Stelzer 2 , J. Busch 2 , A. Imm 1 , S. Schlaubitz 2,3 , B. Lee 3,4 , B. U. Zabel 1 ; 1 Centre for Youth and Adolescence Medicine, Freiburg, Germany, 2 Children’s Hospital of the University of Mainz, Mainz, Germany, 3 Baylor College of Medicine, Houston, TX, United States, 4 Howard Huges Medical Institute, Houston, TX, United States. Skeletogenesis in part takes place in the growth plate where chondrocytes undergo a coordinated and highly regulated process of cell proliferation, differentiation and apoptosis at which stage vascular invasion brings osteoblasts that replace the cartilage extra cellular matrix with trabecular bone. Disruptions to these extremely coordinated processes result in a group of genetic bone diseases. These highly diverse groups of disorders have a complex aetiology. Some of the involved signalling pathways and gene interactions are known but many molecular mechanisms that might explain the pathophysiology of numerous skeletal dysplasias that result from mutations in genes important for normal bone development still remain unknown. While the sequence of the human genome has been completed, many genes are still uncharacterized. Despite the huge number of ESTs in the public domain, there are still genes to be identified in rare tissues that are under-represented. To find cartilage-specific genes a human cartilage cDNA library was generated and 5000 ESTs are sequenced, computationally characterized. We and others have already characterized novel and tissue specific genes. Based on in silico motif searches 49 putative transcription factors have been identified. The temporal and spatial expression patterns of these transcription factors will be determined via in situ hybridization. Those transcription factors that are predominantly expressed in developing cartilage will be further characterized to elucidate their role during cartilage and bone development. This way we hope to identify new genes that are important for bone development and deregulation of those genes might be responsible for causing skeletal dysplasias. P1232. The human CDK5R1 3‘UTR contains distinct subregions affecting transcript stability S. Moncini 1 , M. Venturin 1 , A. Bevilacqua 2 , A. Ratti 3,4 , A. Nicolin 2 , P. Riva 1 ; 1 Department of Biology and Genetics, Medical Faculty, University of Milan, Milan, Italy, 2 Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Milan, Italy, 3 Department of Neurological Sciences, University of Milan, Milan, Italy, 4 Department of Neuroscience, `Dino Ferrari’ Centre, University of Milan-IRCCS Istituto Auxologico Italiano, Cusano Milanino (MI), Italy. Human CDK5R1 encodes for p35, a neurone-specific activator of CDK5, which is involved in neuronal migration and differentiation during CNS development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3’UTR suggests a role of this region in the control of CDK5R1 expression by post-transcriptional regulatory elements modulating mRNA stability or translation efficiency. Bioinformatic analysis showed a high conservation degree in mammals and predicted several AU-Rich Elements (AREs). CDK5R1 3’UTR was cloned in pGL4.71 at the 3’ end of the Renilla luciferase reporter gene to perform Dual Luciferase assays: the construct showed a decreased luciferase activity in six transfected cell lines. The quantitative analysis of luciferase mRNA suggests that CDK5R1 3’UTR affects mRNA stability. We identified five 3’UTR subregions reducing the luciferase activity in some instance with a cell line-dependent way. A region showed a significantly low halflife, suggesting an accelerated mRNA degradation. We also identified, by deletion analysis, a type I ARE displaying a stabilizing effect in two neuroblastoma cell lines. Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3’UTR. We are now attempting to identify, by REMSA and immunoprecipitation assays, stabilizing neuronal proteins that specifically bind the type I ARE, with the final aim of verifying the functionality of this element. The fine tuning of CDK5R1 expression by 3’UTR may play a role in CNS development and functioning, with potential implications in neurodegenerative and cognitive disorders. P1233. Cells to Ct: Direct gene expression analysis from cell lysate J. F. Stevens1 , S. Dong1 , C. Liu1 , R. Fekete2 , A. Nguyen2 , L. Chapman2 ; 1 2 Applied Biosystems, Foster City, CA, United States, Applied Biosystems, Austin, TX, United States. Real-Time reverse transcription (RT)-PCR is a robust, simple and quantitative method for gene expression analysis in biological samples. Traditionally, RNA has been isolated from the sample to remove genomic DNA, RNases, and reverse transcriptase inhibitors before being used as substrate for RT-PCR. RNA isolation is fairly time-consuming and it can lead to loss of RNA in flow-through or incomplete elution with small samples. Direct cell-to-Ct gene expression analysis enables reverse transcription and real-time PCR analysis of RNA from 10-105 cultured cells without RNA isolation or purification. By eliminating the RNA isolation step, gene expression analysis of cultured cell line is substantially expedited and simplified. The unique Cells-to Ct lysis procedure allows the concurrent preparation of cell lysate and removal of genomic DNA in less than ten minute. The lysis procedure takes place at room temperature, allowing simple scale-up to robotic platforms for high-throughput applications. P1234. Theoretical and functional analysis of expressing region of ceruloplasmin pseudogene S. Klotchenko 1 , N. Tsymbalenko 1 , N. Platonova 1 , L. Puchkova 2 ; 1 Research Institute for Experimental Medicine, St. Petersburg, Russian Federation, 2 St. Petersburg State Polytechnic University, St. Petersburg, Russian Federation. Previously using computer analysis we showed that human (NG_ 001106) and chimpanzee (XM_516813) pseudogene of ceruloplasmin (pCp) contain region encoded Cp-like protein. The MitoProt program predicted that putative protein consists from N-terminus (66 aa) corresponding to signal peptide for mitochondrial protein import (SPMPI). Remaining region corresponds to domains 5 and 6 of blood Cp. In HepG2 and HuTu80 cells, pCp transcripts were detected by RT-PCR. pCp polypeptide was found in mitochondrial matrix by Western-blot. To check the biological significance of predicted SPMPI-sequence it was cloned in vectors pEGFP-N3. HEK293 cells were transfected with pEGFP-N3-pCpSPMPI construct. The confocal microscopy data 0
Genomics, technology, bioinformatics showed that GFP was localized in the same intracellular compartments as Mito-dsRed. Also HepG2 cells were transfected with this construct. The cells were collected and subcellular fractions were isolated by differential centrifugation. Western-blot analysis with antibodies to GFP revealed GFP in mitochondria. Population comparative analysis of Cp gene 5’-UTR and translated pCp region in DNA isolated from 117 human blood samples by PCR with following PRLF analysis using of restriction enzymes set was carried out. The insertions or deletions as well as mutations in restriction sites in pCp gene amplicons were not found. At the same time a lot of changes were detected in Cp gene promoter. These data suggest conservatism of pCP region. In rat, expression of mitochondrial Cp has tissue specific pattern, not expresses in newborn and adult animals with copper deficiency, induced by Agfeed. The biological role of putative protein product of pCp in copper metabolism is discussed. P1235. Functional interactions of conserved non-coding (CNCs) sequences with other CNCs using circular chromosome conformation capture (4C) D. Robyr, G. Duriaux-Sail, S. Polti, C. Wyss, S. Deutsch, S. E. Antonarakis; University of Geneva Medical School, Geneva, Switzerland. The comparison of human chromosome 21 (Hsa21) with the mouse syntenic regions led to the identification of roughly 3500 regions displaying an identity of >70% over a length of a least 100 nucleotides of ungaped alignment. About 65% (~ 2300) of these are conserved non-coding sequences (CNCs). Very little is known about the function of most CNCs. We speculated that a functional CNC would interact with its genomic target (i.e. an enhancer would bind to it’s cognate gene promoter). Thus, the identification of any part of the genome that interacts directly with a CNC could provide clues on the function of the latter. We have generated libraries of CNC-interacting DpnII fragments by circular chromosome conformation capture (4C) whose identity is determined by subsequent sequencing. We have generated initial results concerning crosslinking of 18 Hsa21 CNCs with DNA fragments mapping hundreds of kilobases away from the “bait” on the same chromosome, or with fragments on other chromosomes. A total of 87 such potentially interacting DpnII DNA fragments have been identified. Interestingly, the median distance from the cloned DpnII fragments to the nearest conserved region is 661bp with a pvalue < 0.0003 when compared to the distribution of the median of the distances of 3000 random samples of 87 fragments. These results provide initial evidence that the function of CNCs is mediated by their interaction with other conserved regions. P1236. EM algorithm for gene copy number estimation using TaqMan® Assays C. Barbacioru, K. Li, R. Samaha, K. Lazaruk; Applied Biosystems, Foster City, CA, United States. Recently, multiple studies have discovered an abundance of copy number variation of DNA segments ranging from kilobases (kb) to megabases (Mb) in size. Copy number variations can cause disease, as in microdeletion or microduplication disorders, or confer risk to complex disease traits such as HIV-1 infection and glomerulonephritis. TaqMan® gene copy number assays have been developed for accurate detection of genetic variation at gene level using primers and probes designed for genomic DNA. Each well is duplexed with two assays, a FAM dye-based assay designed to detect the genes-ofinterest and a VIC® dye-based assay for reference gene. In this study, we present an algorithm for gene copy number estimation based on EM algorithm for mixtures of normal distributions. The algorithm finds maximum likelihood estimates of parameters in probabilistic models, where the model depends on unobserved samples copy number of the gene-of-interest. Under current protocols, we are capable of distinguishing up to 8 copies of the gene of interest with at least 95% confidence. To evaluate this algorithm, we present experimental results for 5 important drug metabolism genes (CYP2D6, CYP2E1, CYP2A6, GSTM1 and GSTT1) on 270 individual samples from International HAPMAP Project representing 4 different populations. Copy number analysis for these genes shows perfect consistency for sample duplicates. Copy number variations are observed for these genes, with significant differences between these populations. Furthermore, combining this data with SNP data, we demonstrate that departures from diploidy can cause apparent genotyping failure and give inaccurate genotyping. P1237. A computational method to test for genetic relatedness in unrelated individuals L. Xumerle, G. Malerba, P. F. Pignatti; Biology and Genetics, Verona, Italy. Discovering susceptibility loci in complex disease requires a large number of individuals characterized by several thousand markers. An association between gene and disease may be incorrectly estimated if the allele frequencies differ among cases and controls depending on factors other than gene-phenotype correlation, i.e. inbreeding, genotyping errors, unrecognized population stratification, etc. We present a computational method - available in the Jenoware library (http://medgen.univr.it/jenoware/) - designed to compute the probability of genetic relatedness in pairs of individuals. The program computes the likelihood of a pair of individuals to be unrelated over the likelihood to be relatives of I, or II degree respectively, conditional to the genotype. The program works with microsatellites and/or SNPs. False positive rate and power were assessed by simulation in unrelated individuals and in pedigrees. E.g. in order to estimate the support for I degree relatedness (power 80%, and false positive 5%), 10 microsatellites having heterozygosity >= 70% or 25 SNP having heterozygosity >= 15% are needed. We are extending the method to associated DNA markers that can be included as inferred haplotypes in the models to be tested. P1238. Genome-wide analysis of copy number variations in patients with mental retardation by single nucleotide polymorphism arrays J. Wagenstaller 1 , S. Spranger 2 , B. Lorenz-Depiereux 1 , B. Kazmierczak 2 , M. Nathrath 3 , D. Wahl 4 , B. Heye 5 , D. Gläser 6 , V. Liebscher 7 , T. Meitinger 1,5 , T. M. Strom 1,5 ; 1 GSF - National Research Center, Institute of Human Genetics, Munich, Germany, 2 Praxis für Humangenetik, Bremen, Germany, 3 Technical University, Department of Pediatrics, Munich, Germany, 4 Praxis für humangenetische Beratung, Augsburg, Germany, 5 Technical University, Institute of Human Genetics, Munich, Germany, 6 Zentrum für Humangenetik, Neu-Ulm, Germany, 7 University of Greifswald, Institute of Mathematics, Munich, Germany. We investigated 67 children with unexplained mental retardation who had additional, but often mild symptoms. All children had inconspicuous high resolution banding analysis. The DNAs were analyzed using the Affymetrix GeneChip 100K arrays. Data analysis was performed with median normalization and genotype-specific dosage calculation using R-scripts and revealed 12 copy number variations (CNVs), 9 deletions and 3 duplications, that are most likely causative for mental retardation because they either arose de novo or are larger than known polymorphisms. One of those was a maternally inherited 1.4 Mb duplication in Xp22.31 in a male patient which includes the STS gene. The chromosome carrying the duplication was non-randomly inactivated in the mother. Two of the CNVs were de novo deletions affecting only a single gene. All CNVs were confirmed by quantitative PCR. They varied in size from 200 kb to 8 Mb. Five of the CNVs were flanked by low-copy number repeats. Three of them were known microdeletion syndromes. The fourth one is a deletion on chromosome 15q25.2 that was not described before. The fifth one is the duplication in Xp22.31. We compared different array types. The signal-to-noise ratio (SNR) of the 500K Affymetrix array was lower than the SNR of the 100K Affymetrix array. The SNR of the 300K Illumina arrays was in between. The increase of the number of features and a new design should improve the resolution and allow the reliable detection of gain and losses of single genes. P1239. Multiplex Amplicon Quantification, a novel method for PCR based, high-throughput copy number variation analysis. D. Goossens 1,2 , P. De Rijk 1,2 , L. Heyrman 1,2 , B. Harding 3,2 , W. Glassee 3,2 , J. Del-Favero 1,2 ; 1 Applied Molecular Genomics Group, Department of Molecular Genetics, VIB, Antwerp, Belgium, 2 University of Antwerp, Antwerp, Belgium, 3 Department of Molecular Genetics, VIB, Antwerp, Belgium. Recently, a striking abundance of copy number variation (CNV) was discovered throughout the human genome. Although it has been sug- 0
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Volume 15 Supplement 1 June 2007 ww
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Table of Contents Spoken Presentati
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Plenary Lectures Plenary Lectures P
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Plenary Lectures labile iron can be
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Concurrent Symposia S07. Vertebrate
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Concurrent Symposia S17. Towards a
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Concurrent Symposia 11 at E13.5 sim
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Concurrent Symposia 1 phomagenesis.
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cover cryptic mutations in Drosophi
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Concurrent Sessions first excluded
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Concurrent Sessions of 210 ancestry
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Concurrent Sessions C23. Literature
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Concurrent Sessions a boy with a ty
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Concurrent Sessions C40. Mutation o
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Concurrent Sessions C48. CHROMSCAN:
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Concurrent Sessions C57. RNAi-based
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Concurrent Sessions been replicated
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Concurrent Sessions involved in an
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Concurrent Sessions tion considers
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Clinical genetics lateral neurosens
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Clinical genetics apparently sporad
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Clinical genetics itar syndrome, wh
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Clinical genetics diseases, Foundat
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Clinical genetics P0041. The first
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Clinical genetics EFNB1 was found t
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Clinical genetics of the CSB gene.
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Clinical genetics growth retardatio
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Clinical genetics PCR with a modifi
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Clinical genetics pound heterozygou
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Clinical genetics plicated: two cou
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Clinical genetics P0105. APC gene m
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Clinical genetics an indication of
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Clinical genetics great importance
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Clinical genetics P0133. Hidrotic e
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Clinical genetics eight patients as
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Clinical genetics P0152. Kabuki syn
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Clinical genetics P0161. Intrafamil
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Clinical genetics matter and normal
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Clinical genetics type at 550 bands
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Clinical genetics will be confirmed
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Clinical genetics nosed. Accurate r
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Clinical genetics which has not bee
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Clinical genetics P0217. Partial mo
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Clinical genetics fested progeroid
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Clinical genetics A 29 years old ma
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Clinical genetics ing loss (HHL). I
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Clinical genetics dació Son Llatze
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Clinical genetics These preliminary
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Clinical genetics P0272. Williams S
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Cytogenetics Po02. Cytogenetics P02
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Cytogenetics to reports from Saudi
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Cytogenetics P0298. Female-specific
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Cytogenetics P0306. Lipoatrophic pa
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Cytogenetics 22 leading to the form
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Cytogenetics somal sperm and that o
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Cytogenetics University of Belgrade
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Cytogenetics Within the same metaph
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Cytogenetics Less than 10 cases of
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Cytogenetics lar markers taken from
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Cytogenetics Brain Unaffected Schiz
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Cytogenetics ability with no speech
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Cytogenetics P0389. An age based cy
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Cytogenetics P0399. Molecular Chara
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Cytogenetics ing of complex examina
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Cytogenetics letion in 5q33 in all
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Cytogenetics and his son carried th
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Prenatal diagnosis tion about famil
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Prenatal diagnosis P0443. The risk
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Prenatal diagnosis in house PCR pro
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Prenatal diagnosis P0462. Increased
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Prenatal diagnosis P0471. Preimplan
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Prenatal diagnosis agreement with w
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Prenatal diagnosis of Turner Syndro
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Cancer genetics ously defined for d
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Cancer genetics Their frequencies w
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Cancer genetics tion Clinic-Portugu
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Cancer genetics tric carcinogenesis
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Cancer genetics P0532. Secondary ch
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Cancer genetics P0542. Differential
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Cancer genetics gastric cancer risk
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Cancer genetics ania, 3 The Institu
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Cancer genetics low: 8% (8/98 sampl
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Cancer genetics P0578. Somatic mito
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Cancer genetics P0587. Differential
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Cancer genetics cinoma (ESCC), whic
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Cancer genetics method to measure t
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Cancer genetics pression (compared
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Cancer genetics to available biolog
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Molecular and biochemical basis of
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Genetic analysis, linkage, and asso
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Page 353 and 354: Author Index 1 Arslan-Krichner, M.:
Page 355 and 356: Author Index Borkowska, A.: P1089 B
Page 357 and 358: Author Index Coviello, D.: P0078, P
Page 359 and 360: Author Index Estivill, X.: P1216, P
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Author Index Graf, S. A.: P0021 Gra
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Author Index 1 Josifiova, D.: PL07
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Author Index Laurier, V.: C03 Lauri
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Author Index Melo, D. G.: P0260, P0
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Author Index Osorio, P.: P0218 Osta
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Author Index Reese, T.: C06 Regal,
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Author Index 1 Shaposhnikov, S.: P0
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Author Index Touitou, I.: P0733 Tou
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Author Index Xiao, C.: P1241 Xie, G
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Keyword Index ARMR: P0907 array CGH
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Keyword Index Consanguineous marria
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Keyword Index 1 Fronto-temporal dem
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Keyword Index IRF5: P1086 IRF6: P07
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Keyword Index NBS1 gene: P0567 NBS-
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Keyword Index P1085 Rep1: P1104 rep
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Keyword Index vision: P0632 Vitamin
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Notes 1
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A natural choice in Fabry Disease P
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