Day 2 :
Keynote Forum
Kathleen S Wilson
UT south Western Medical Center, USA
Keynote: The application of validation and proficiency testing concepts from current clinical genetic diagnostics for the implementation of new genetic technologies Kathleen S Wilson, UT south
Time : 10:00-10:30
Biography:
Kathleen S Wilson is a Professor of Genetics in the Department of Pathology and the McDermott Center for Human Growth and Development at the University of Texas Southwestern Medical Center in Dallas, Texas. She is board-certifi ed by the American Board of Pathology (ABP) in Clinical Pathology and the AmericanrnBoard of Medical Genetics (ABMG) in Clinical Cytogenetics. She is a fellow member of the College of American Pathologists (CAP) and the American Collegernof Medical Genetics (ACMG). She is currently the Director of the Cytogenomic Microarray Analysis (CMA) laboratory at U.T. Southwestern, Course Director forrnthe medical student Genetics and Pathology Courses, member of the U.T. Southwestern Medical School Admissions Committee and Chair of the DepartmentalrnPromotions and Tenure Committee.
Abstract:
The advent of new genetic technologies that may be utilized for clinical diagnostics is rapid and the actual mechanics of howrnthese evolving technologies are implemented in a clinical laboratory can be challenging. Participation for over a decade in therndevelopment of regulatory guidelines and profi ciency testing through the College of American Pathologists/American Collegernof Medical Genetics Cytogenetics Resource Committee as well as experience in the Genetic Diagnostics Laboratories at U.T.rnSouthwestern have elucidated key concepts that were critical in the successful implementation of the technologies of fl uorescencernin situ hybridization (FISH) and cytogenomic microarray analysis (CMA) into clinical practice, and now have relevance for thernrapidly evolving sequencing technologies. Th is presentation will discuss these concepts and give specifi c examples of how thernlessons learned from prior technology have relevance for the clinical genetic diagnostics of the future.
Keynote Forum
Mariusz Grzelakowski
Applied Biomimetic, USA
Keynote: Biomimetic membranes
Time : 10:30-11:00
Biography:
Mariusz Grzelakowski is Chief Scientific Officer at Applied Biomimetic. Before joining Applied Biomimetic in 2011 Mariusz has held positions at NanoH2O in Los Angeles, Novatis Pharma in Basel, and Zachem in Bydgoszcz in Poland. rnMariusz Grzelakowski holds a Ph.D. in Chemistry from the University of Basel (2009), and M.Sc. and B.Sc in Chemistry from Nicholas Copernicus University, Toruń, Poland (2002 and 2000 respectively).rn
Abstract:
Genetically engineered membrane proteins such as aquaporins, incorporated into synthetic membranes, are considered a promising biomimetic separation technology and have been intensively studied over the last few years. The most common strategies to synthesize Aquaporin Biomimetic Membranes (ABM) are to deposit the aquaporin incorporated lipid or block copolymer vesicles onto porous substrates or to integrate them within the active layer of polyamide membranes. However, ABMs with orders of magnitude improvement in permeability and perfect salt rejections proposed in initial work have not been realized. Early results were based on materials and methods that were rudimentary, especially considering the progress that has been made in this field. We have conducted a comprehensive evaluation of the true promise of these membranes using improved methods for protein expression and purification, polymer synthesis, self-assembly, experimental evaluation as well as calculations that more directly compare the outcome of biophysical evaluations to those used in the separations membrane industry. We propose these as standard methods for use in ABM research. We further describe an example of improved techniques and methodology in application to colloidal coating of aquaporin enabled separations membranes. Development activities at Applied Biomimetics Inc. focused on incorporation of membrane proteins polymer vesicles and development of a coating platform using colloidal particles as building blocks.rn rnBlock-co-polymer vesicles have shown to be a versatile coating platform allowing for utilization of membrane protein function in the form of flat-sheet membrane. Membranes characterized by a wide range of molecular cut-offs were coated, and pure water flow was regulated by the amount of incorporated protein. This approach allowed for a 3-4-fold increase in flow over existing commercial membranes at a wide range of molecular cut-offs with modest aquaporin levels. rn
- Track 2: Functional Genomics Track 5: Pharmacogenomics & Personlaized Medicine
Chair
Luciano Brocchieri
University of Florida, USA
Co-Chair
John M Rosenfeld
EMD-Millipore, USA
Session Introduction
John M Rosenfeld
EMD-Millipore, USA
Title: Genomics and chromatin analysis methods for profiling protein, RNA and DNA interactions in chromatin
Biography:
John M Rosenfeld received his BS from Georgetown University, and received his PhD in molecular biology & biochemistry from the University of California, Irvine. He performed his Post-Doctoral training in the laboratory of Dr Ronald Evans at the Salk Institute on the topic of profi ling transcriptional targets of orphan nuclear receptors. He joined Merck-Millipore in 2003, and has been developing research tools to explore gene regulation for the past 11 years. In addition to developing research tools and assays for epigenetic analysis, he is now responsible for managing platform technology development and external innovation for EMD Millipore and is part of the bioscience scientifi c networking group in this company.
Abstract:
Chromatin immune-precipitation provides an in vivo picture of protein association within the dynamic cellular chromatin environment. Over the past decade, additional resolution on chromatin structure has been elucidated using other techniques that capture proteins or nucleic acids to uncover the composition of the components of chromatin, including regulatory proteins, modifi ed histones, modifi ed genomic DNA and the new putative chromatin regulatory molecule, non-coding RNAs. Th ese precipitation techniques, both immune based as well as nucleic acid capture based, allow dissection of the role of these molecules in establishing and characterizing chromatin state. Th e methods can be easily combined with current library construction techniques to provide genome wide views of these interactions under experimental treatments & genetic backgrounds. Th e information provided by ChIP (Chromatin Immuno-Precipitation), Nuclear RIP (RNA Binding Protein Immuno-Precipitation of chromatin) and ChIRP (Chromatin Isolation by RNA ) will be discussed.
Maarten Rudolph Leerkes
Medical Science & Computing Inc., USA
Title: Discovery of novel molecular targets in SIV infected non-human primates serve as models for human HIV drug targeting
Biography:
Maarten Rudolph Leerkes is an accomplished Bioinformatics Scientist at NIH-BCBB (Bioinformatics and Computational Biosciences Branch). He works on developing novel quantitative biology methods. He has worked on identifying molecular signatures for disease prognosis and treatment prediction in patient sub-populations in biotech industry as well as on product development including study design for product validation in clinical settings. His PhD, Post-doctoral and research experiences span academia as well as biotech industry settings where he focused on the use of bioinformatics to interpret sequencing data (NGS) and to fi nd patterns that can be extrapolated into diagnostic tools for improving treatment for patients.
Abstract:
We have analyzed RNAseq data in the context of re-annotating the genome of African green Monkeys (AGM). RNA-seq experiments were designed to answer questions regarding the mechanisms underlying the lack of disease progression in these natural Simian Immunodefi ciency Virus (SIV) hosts which are still poorly understood. Although the AGM genome is available, it is poorly annotated. Th e rhesus genome is also available for dual-genome predictions and although it has its own limitations, it can be used in combination with RNA-seq data to re-annotate the AGM genome. Th e patas monkey is an interesting third primate study model but does not have a reference genome available and depends on de novo assembly of RNA-seq reads to be analyzed. De novo assembly compared with genome-based splicing detection helps cross-validate methods. Cross-species comparisons can shed light in more detail on resistance mechanisms related to SIV and HIV infections. RNA-seq studies focusing on splicing signatures are an essential tool for both genome re-annotation and biomarker discovery. As such, this is of interest for upcoming RNA-seq studies with the objective to more accurately defi ne how specifi c splicing signatures render African green monkeys resistant to progressive SIVagm infection. Th is is promising in the discovery of novel molecular targets in the process of SIV infection and can serve as a model for human HIV targets and thus serves as a compelling example that impacts genomic advances on global health.
Huijuan Wang
Northwest University, China
Title: Rapid and reliable genotyping of HLA-B*58:01 in four Chinese populations using a single-tube duplex real-time PCR assay
Biography:
Huijuan Wang has graduated from Northwest University of China and received her PhD degree in Biochemistry and Molecular biology. Since 2011, she was recruited as a Teaching Staff by College of Life Science, Northwest University mainly focuses on the clinical application of pharmacogenomics fi ndings in disease treatment especially cancers including development of genotyping methods and reagents for drug-related biomarkers and mechanic study of cancer-related biomarkers. Besides, lots of efforts are also dedicated on the study of the molecular mechanism underlying the resistance of anticancer drugs such as BRAF inhibitors and endocrine therapy.
Abstract:
Aims: HLA-B*58:01 is strongly associated with allopurinol induced Severe Cutaneous Adverse Reactions (SCARs). Th e aim of this study was to develop a new, convenient and economical method for HLA-B*58:01 genotyping and to investigate the distribution of HLA-B*58:01 in diff erent Chinese populations. Methods: Combined with ARMS (Amplifi cation Refractory Mutation System) primers and TaqMan probe, a single-tube duplex real-time PCR assay for HLA-B*58:01 typing was established with ACTB as an internal control. Using this method, the prevalence of HLA-B*58:01 in 349 samples including 100 Northern Chinese Han, 100 Buyei, 99 Tibetan and 50 Uighur were determined. Th e reliability and specifi city was assessed by comparison of genotyping results in 100 Buyei samples with Sequence-Based Typing (SBT). Meanwhile, the linkage status of rs9263726 in PSORS1C2 with HLA-B*58:01 in four Chinese populations was analyzed. Results: Th e HLA-B*58:01 genotyping result in 100 Buyei samples by real-time PCR was in 100% concordance with SBT and the detecting limit of this assay was 50 pg. Th e frequency of the HLA-B*58:01 allele in Buyei minority (17%) was signifi cantly higher than that in Han (4%), Tibetan (5.1%) and Uighur (2%) populations (p<0.05). Th e complete linkage of HLA-B*58:01 with SNP rs9263726 previously reported in a Japanese population was not observed in the Chinese populations. Conclusion: Th e newly developed assay proves to be rapid, cost-eff ective and reliable for HLA-B*58:01 detection prior to allopurinol administration. Meanwhile, the rs9263726 could not be used as an alternative marker to HLA-B*58:01 in clinical diagnosis for allopurinol-induced SCAR especially in Chinese populations.
Luciano Brocchieri
University of Florida, USA
Title: Translational evidence and the accuracy of prokaryotic gene annotation
Biography:
Luciano Brocchieri has completed his PhD in evolutionary biology at the Unversity of Parma (Italy) in 1992 and Postdoctoral studies at Stanford University, Department of Mathematics, in 1996. He was Senior Scientist at the Math Department in Stanford University and is currently Assistant Professor at the Department of Molecular Genetics and Microbiology, University of Florida. He has been invited at several international conferences and has published about fi fty papers in highly reputed international scientifi c journals. He is best known for his work in computational sequence analysis, protein evolution, and gene identification.
Abstract:
Gene annotation in prokaryotic genomes is challenged by inconsistencies among gene predictors and by incomplete or ambiguous information from evolutionary conservation, and recent analyses have evidenced the existence of genes missing from 1000 genome annotations. Popular gene prediction methods predict in the same genomes almost 20% more genes that are not in the annotations. Although for the majority of the excluded genes there is no convincing evidence of conservation, justifying their exclusion from annotations, we identifi ed among excluded genes a substantial fraction of genes conserved in sequence and in length across genera or phyla, many of which are corroborated by all methods. Introducing the N-PACT (N-Profi le Analysis Computational Tool) methods, we found that several of these ORFs could also be identifi ed by their signifi cant compositional periodicity. From information from gene prediction, evolutionary conservation and sequence 3-base periodicity, we estimated that as much as 30% of the currently annotated genomic inter-genic space could be occupied by unrecognized coding regions. We verifi ed expression of predicted genes in Pseudomonas aeruginosa PAO1 by RIBO-seq analysis, the genome-wide sequencing of mRNA regions undergoing translation by ribosome foot-printing. From the analysis of ribosome-footprint distributions, we could confi rm expression of the majoirity of the annotated genes, provide evidence of some mis-predicted starts-of-translation, and verifi ed expression of many predicted genes not included in the published annotation. Furthermore, ribosome footprints provided evidence of the existence of coding regions not predicted by any method, of previously unrecognized mechanisms of post-transcriptional regulation of translation (‘leader peptides’), as well as of alternative translational products.
Alexander Kaplun
QIAGEN Bioinformatics, USA
Title: PGMD: A comprehensive pharmacogenomic database for personalized medicine and drug discovery
Biography:
Alexander Kaplun has completed his PhD from Ben Gurion University and Postdoctoral studies from Karmanos Cancer Institute. He is a Senior Scientist, Advanced Genomics Integrated Solutions at QIAGEN Bioinformatics, Global Leader in development and distribution of clinical and biological software tools and databases. He has published more than 20 papers in peer-reviewed journals.
Abstract:
The Pharmaco-Genomic Mutation Database (PGMD) is a comprehensive manually curated pharmaco-genomics database. Th e aim of this database is to provide a comprehensive resource for all variants that have been reported to have a pharmacogenomic eff ect in human studies and to describe those variants by exact genomic location and sequence alterations for application to NGS data analysis. Th e database is designed to contain extensive information as evidence for these associations including provenance of every observation. Two major sources of PGMD data are peer reviewed literature and FDA drug labels. PGMD curators capture information on exact genomic location and sequence changes resulting phenotype, drugs administered, patient population, study design, disease context, statistical signifi cance and other properties of reported pharmaco-genomic variants. Variants are annotated into functional categories basing on their infl uence on pharmacokinetics, pharmacodynamics, effi cacy or clinical outcome. Th e current release of PGMD includes nearly 140000 unique pharmaco-genomic observations, covering all 24 disease super classes and 1377 drugs. Over 2800 genes have associated pharmaco-genomic variants including genes in proximity to intergenic variants. PGMD is optimized for use in annotating next generation sequencing data by providing genomic coordinates for all covered variants including SNPs, insertions, deletions, haplotypes, diplotypes, VNTRs, copy number variations and structural variations.
Haidan Mostafa Salem
Cairo University, Egypt
Title: Possible mutagenicity in Ha-Ras gene by TiO2 nanoparticles in mice
Biography:
Haidan M Salem is an Academic Lecturer of Molecular Biology and Genetics and completed her PhD in 2014 at Cairo University, Egypt. Currently, she is supervising one PhD thesis and four MSc theses, working on two projects entitled "Investigation of the possible role of par-4 as a therapeutic pro-apoptotic protein in hepatocellular carcinoma" and "Effi ciency of using Camptothecin and 7-hydroxy-6-methoxycoumarin encapsulated in chitosan nanoparticles in Hepatocellular carcinoma treatment”. She has taught a number of biology courses, a number of practical molecular and cytogenetic assays and also has an extensive experience working with DNA, RNA and proteins.
Abstract:
Increase using of nanoparticles in the industry ranging from health care products to cosmetics to dietary supplements let human exposure to nanoparticles is a driving concern. TiO2 nanoparticles are used in a broad range of applications due to their high stability, corrosion resistance and photocatalytic properties. Recent evidences have shown TiO2 nanoparticles to induce inflammatory and genotoxic response in diff erent animal and human cell lines. However, the mechanisms involved in nano- TiO2 induced genotoxicity and carcinogenicity have not been clearly defi ned and are poorly studied in vivo. Ras gene is a protooncogene that normally regulate the cell proliferation, the aim of the present study is to evaluate point mutation that may be induced by diff erent treatments (acute and sub-acute) and doses of titanium dioxide nanoparticles TiO2 (<100 nm) in testis, lung and kidney of mice in Ha-ras gene exons 2 and 3 (hot spot exons) as an example of oncogenes using PCR-Single-Strand Conformation Polymorphism (SSCP) analysis and sequencing of the mutant samples. Sequencing of mutant samples revealed substitution mutations caused amino acid substitution and insertion mutations caused frame shift and insertion mutations found outside coding sequence. In conclusion, Single Strand Conformation Polymorphism (SSCP) analysis of Ha-ras exons 2, 3 and sequencing studies showed that TiO2 nanoparticles signifi cantly increased the incidence of band alterations and induced diff erent point mutations at diff erent dose levels and treatments in lung, kidney and testis compared to control. Anyway, further investigation should be considered to fi gure out the possible role of long exposure of TiO2-NPs in cancer initiation.
Hao Mei
University of Mississippi Medical Center, USA
Title: The uniform-score gene set analysis of genetic pathways associated with diabetes traits
Biography:
Hao Mei has completed his PhD from North Carolina State University with majors in Statistics and Bioinformatics and his Postdoctoral studies from Center for Human Genetics at Duke University. He is currently Associate Professor of University of Mississippi Medical Center and Professor of Shanghai Jiao Tong University. He is an Active Investigator of Jackson Heart Study and Atherosclerosis Risk in Communities Study and he has published more than 20 papers in reputed journals for genetic study of complex disease.
Abstract:
Genetic heritability and expression study have shown that diff erent diabetes traits have common genetic components and pathways. Th e Uniform-Score Gene-Set Analysis (USGSA) is a computationally effi cient method for pathway enrichment test that unifi es diff erent gene measures by a uniform score for identifying pathways from genome-wide association and expression data and an R package of snp Gene Sets is implemented to facilitate the analysis. USGSA was applied to identify common pathways associated with diabetes traits based on public dbGaP GWAS results following a two-stage study strategy: the stage I of 11 Framingham Heart Study (FHS) GWAS and the stage II of 5 independent GWAS. Th e study identifi ed 7 gene sets that contain binding motifs at promoter region of component genes for 5 Transcription Factors (TFs) of FOXO4, TCF3, NFAT, VSX1 and POU2F1 and microRNA of mir-218. Th ese gene sets include 25 common genes that are among top 5% of the gene associations over genome for all GWAS. To further evaluate the identifi ed diabetes pathways, 30 microarray data of diff erent tissues was retrieved from the Gene Expression Omnibus. Th e USGSA with meta-analysis showed that 6 gene sets are also enriched for top 5% of the diff erential gene expressions. Th e pathway analysis suggested that diff erent diabetes traits share common pathways and diabetes pathogenesis at varied tissues is potentially regulated by common TFs and microRNA.
Rana Ahmed Youness
German University, Egypt
Title: Enhancing natural killer cytotoxicity by miR-486-5p in hepatocellular carcinoma
Biography:
Rana Ahmed Youness has completed her MSc degree from German University in Cairo (GUC) under the supervision of Professor Dr Ahmed Ihab Abdelaziz, Founder of the Molecular Pathology Research Group (MPRG).
Abstract:
Insulin-like Growth Factor-1 Receptor (IGF-1R) activation is a hallmark in Hepato-Cellular Carcinoma (HCC), stimulating several mitogenic signaling pathways most importantly PI3K/Akt/mTOR pathway. As an essential cornerstone of the innate immune system, Natural Killer (NK) cells are recognized as the fi rst native defender against HCC. Intersentingly, NK cells are among the immune cells with the highest level of IGF-1R and it was also reported to have a prominent role in NK cell differentiation. NK cells are known to be activated by the activating receptor, NKG2D, mediating its cytotoxicity mainly through perforins release. IGF-1R expression was found to be regulated by several miRNAs. However, miR-486-5p has never been investigated in HCC. So, the aim of this study was to control the HCC tumor progression through the direct impact of miR-486-5p on NK cells as well as their target hepatocytes in an immunotherapeutic approach. Huh7 cells were cultured and NK cells were isolated from 27 HCC patients. Both cell types were transfected by miR-486-5p using lipofection. Total RNA was extracted and quantifi ed using qRTPCR. Viability and proliferation analysis were performed using MTT and BrdU assays. miR-486-5p showed signifi cant down regulation of both IGF-1R and its down-stream oncoprotein mTOR in Huh7 cells and consquently, Huh-7 cellular viability and proliferation were repressed. Upon ectopic expression of miR-486-5p in NK cells of HCC patients both NKG2D and perforins expression were signifi cantly elevated. However, a signifi cant down-regulation of IGF-1R and mTOR were observed. In conclusion, it was shown that miR-486-5p has a dual role in enhancing NK cell cytotoxicity and harnessing the tumor progression of its target hepatocyte mainly through tuning essential members of IGF-axis.
Daniel Rotroff
North Carolina State University, USA
Title: Common and rare variant analysis of fibrate drug response in type-2 diabetics in the ACCORD clinical trial
Biography:
Daniel Rotroff is a Post-doctoral Research Scholar working with Motsinger-Reif in the Bioinformatics Research Center at NCSU. He has obtained a Master of Science degree in Public Health in 2010 and a PhD in Environmental Science and Engineering from the University of North Carolina at Chapel Hill in 2013. He has authored 20 publications that have accrued over 800 academic citations and his current research focuses on Assessing in utero epigenetic modifi cation due to maternal smoking, using metabolomics approaches for biomarker discovery and identifying genetic variants associated with drug-response phenotypes.
Abstract:
Cardio-Vascular Disease (CVD) is the leading cause of death worldwide. Individuals with type-2 diabetes are at an increased risk of CVD and alterations in total cholesterol (TC), LDL, HDL and triglycerides (TG) are known risk factors of CVD. Fenofi brate is a commonly prescribed cholesterol lowering drug but there is heterogeneity in treatment response. Here we conduct a genomewide association study to investigate common and rare genetic variants associated with lipid changes in 1261 diabetics from the ACCORD clinical trial for ~90 days of fenofi brate treatment. Analysis was also stratifi ed by white (n=773) and black (n=123) subjects. A total of 26, 35 and 120 common variants, mapping to 5, 7 and 24 genes were associated with change in TC, LDL, HDL and or TG in all races, white and black respectively (p<1×10-6). In addition, 7 genes were associated with changes in TG in the rare variant analysis (q<0.05). Signifi cant genes in the common and rare variant analysis were tested for gene expression changes in mice treated with fenofi brate. Th e validation study found 3 genes in the common variant analysis (SMAD3, ATP13A1 and IPO11) displayed signifi cantly decreased gene expression in mice treated with fenofi brate (q<0.25). RAB27B and DCUN1D4 were signifi cantly associated with TG in the rare variant analysis and displayed signifi cantly decreased and increased gene expression respectively. SMAD3 is an intracellular mediator of TGFβ and SMAD3-KO mice have been known to have increased insulin sensitivity and reduced adiposity. Th ese results may provide new biomarkers for fenofi brate drug response and lead to new therapeutic targets.