Splicy: a web-based tool for the prediction of possible alternative splicing events from Affymetrix probeset data

BACKGROUND: The Affymetrix technology is nowadays a well-established method for the analysis of gene expression profiles in cancer research studies. However, changes in gene expression levels are not the only way to link genes and disease. The existence of gene isoforms specifically linked with cancer or apoptosis is increasingly found in literature. Hence it is of great interest to associate the results of a gene expression study with updated evidences on the transcript structure and its possible variants. RESULTS: We present here a web-based software tool, Splicy, whose primary task is to retrieve data on the mapping of Affymetrix probes to single exons of gene transcripts and displaying graphically this information projected on the gene physical structure.Starting from a list of Affymetrix probesets the program produces a series of graphical displays, each relative to a transcript associated with the gene targeted by a given probe. The information on the transcript-by-transcript and exon-by-exon mapping of probe pairs can be retrieved both graphically and in the form of tab-separated files. The mapping of single probes to NCBI RefSeq or EMBL cDNAs is handled by the ISREC mapping tables used in the CleanEx Expression Reference Database Project. We currently maintain these mappings for most popular human and mouse Affymetrix chips, and Splicy can be queried for matches with human and mouse NCBI RefSeq or EMBL cDNAs. CONCLUSION: Splicy generates probeset annotations and images describing the relation between the single probes and intron/exon structure of the target transcript in all its known variants. We think that Splicy will be useful for giving to the researcher a clearer picture of the possible transcript variants linked with a given gene and an additional view on the interpretation of microarray experiment data. Splicy is publicly available and has been realized in the framework of a bioinformatics grant from the Italian Cancer Research Association

729 inheritable silencing of endogenous gene by hit and run targeted epigenetic editing

Gene silencing holds great promise for the treatment of several diseases and can be exploited to investigate gene function and activity of the regulatory genome. Here, we develop a novel modality of gene silencing that exploits epigenetics to achieve stable and highly efficient repression of target genes. To this end, we generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of chromatin-modifying enzymes involved in silencing process of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains (namely KRAB, DNMT3A and DNMT3L) that, when transiently co-assembled on the promoter of the reporter cassette, recreate a powerful embryonic-specific repressive complex capable of inducing full and long-term (>150 days) silencing of transgene expression in up to 90% of the cells. The ATR-induced silencing was cell type and locus independent, and resistant to metabolic activation of the cells. Importantly, these findings were holding true also for endogenous genes embedded in their natural chromatin context, as shown for the highly and ubiquitously expressed B2M gene. Here, transient co-delivery of TALE-based ATRs resulted in loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of the cells. This phenotype was associated with a drastic switch in the epigenetic and transcriptional state of the constitutively active B2M promoter, which become highly decorated with de novo DNA methylation and deprived of RNAP II. Importantly, silencing was sharply confined to the targeted gene and resistant to INF-γ, a potent natural activator of B2M. We further extended these studies by showing that our silencing approach is portable to the CRISPR/dCas9 DNA binding technology. In this setting, comparable levels of B2M silencing (up to 80%) were achieved using either pools or even individual sgRNAs coupled to dCas9-based ATRs. Yet, adoption of this technology allowed performing simultaneous, highly efficient multiplex gene silencing within the same cell, as shown for B2M, IFNAR1 and VEGFA. Finally, we assessed resistance of the silenced gene to activity of potent artificial transcription activators and chromatin remodelers, and found that only targeted DNA demethylation was able to reawaken the silent gene. This allowed performing iterative cycles of silencing and reactivation of the same gene in the same cell population. Overall, these data provide the first demonstration of efficient and stable epigenetic silencing of endogenous genes upon transient delivery of ATRs. This was accomplished by repurposing the ERVs silencing machinery, which instructs self-sustaining repressive epigenetic states to the target gene. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for both basic and translational research

36. Genome-Wide Insight Into the Transcriptional Modulations Triggered By Lentiviral Transduction in Human Hematopoietic Stem Cells

Recent studies suggest that hematopoietic stem cells (HSC) can sense foreign nucleic acids and pathogen-associated molecular patterns (PAMPs). Exposure to lentiviral vectors (LV) upon gene transfer may thus trigger acute host responses in HSC that could potentially impact on their biological properties, although no comprehensive studies are available to date. We have performed a high throughput RNA-Seq analysis on human cord-blood (CB)-derived CD34+ hematopoietic stem and progenitor cells (HSPC) exposed to research- or clinical-grade VSV-g pseudotyped (SIN) LV at a high multiplicity of infection, matching current clinical vector dose requirements. As controls, cells were exposed to non-transducing Env-less, genome-less or heat inactivated control vectors or kept in culture untreated. RNA was extracted at different times early after transduction, processed and ran in Illumina HiSeq2000. Analysis of Differential Expression in Time Course was performed using LIMMA R/BioConductor library. Key pathways were assessed by Term Enrichment Analysis considering KEGG pathways and Gene Ontology Biological processes. Transduction with both research-and clinical-grade LV significantly triggered DNA damage and apoptosis-related responses. In particular, p53 signaling was among the most significantly altered pathways (p<3.47×10−14) and induction of several key players, including a 8-fold increase in p21 mRNA, was further confirmed by Taqman. This signaling occurred also in bone-marrow-derived CD34+ cells and was integration-independent as Integrase-Defective LV (IDLV) induced p21 to a similar extent as LV. Furthermore, equal induction was observed in all CD34+ subpopulations, including in the most primitive CD38-CD133+ fraction. Finally, LV/IDLV exposure lead to a slight but significant increase in the percentage of apoptotic HSPC in culture (p<0.001) as compared to control vector exposed cells and untreated controls. Experiments are ongoing to further investigate the potential short and long-term consequences of this signaling on the biological properties of HSPC in vitro and in vivo. Overall, our results suggest for the first time that LV transduction triggers transcriptional changes in HSPC involving pathways pivotal for their biology. Better understanding of the potential functional consequences this may have will be important for the development of improved gene therapy protocols

531. Computational Pipeline for the Identification of Integration Sites and Novel Method for the Quantification of Clone Sizes in Clonal Tracking Studies

Gene-corrected cells in Gene Therapy (GT) treated patients can be tracked in vivo by means of vector integration site (IS) analysis, since each engineered clone becomes univocally and stably marked by an individual IS. As the proper IS identification and quantification is crucial to accurately perform clonal tracking studies, we designed a customizable and tailored pipeline to analyze LAM-PCR amplicons sequenced by Illumina MiSeq/HiSeq technology. The sequencing data are initially processed through a series of quality filters and cleaned from vector and Linker Cassette (LC) sequences with customizable settings. Demultiplexing is then performed according to the recognition of specific barcodes combination used upon library preparation and the sequences are aligned to the reference genome. Importantly, the human genome assembly Hg19 is composed of 93 contigs, among which the mitochondrial genome, unlocalized and unplaced contigs and some alternative haplotypes of chr6. While previous approaches aligned IS sequences only to the standard 24 human chromosomes, using the whole assembled genome allowed improving alignment accuracy and concomitantly increased the amount of detectable ISs. To date, we have processed 28 independent human sample sets retrieving 260,994 ISs from 189,270,566 sequencing reads. Although, sequencing read counts at each IS have been widely used to estimate the relative IS abundance, this method carries inherent accuracy constraints due to the rounds of exponential amplification required by LAM-PCR that might generate unbalances on the original clonal representation. More recently, a method based on genomic sonication has been proposed exploiting shear site counts to tag the number of original fragments belonging to each IS before PCR amplification. However, the number of cells composing a given clone could far exceed the number of fragments of different lengths that can be generated upon fragmentation in proximity of that given IS. This would rapidly saturate the available diversity of shear sites and progressively generate more and more same-site shearing on independent genomes. In order to overcome the described biases and reliably quantify ISs, we designed and tested a new LC encoding random barcodes. The new LC is composed of a known sequence of 29nt used as binding site for the primers upon amplification steps, a 6nt-random barcode, a fixed-anchor sequence of 6nt, a second 6nt-random barcode and a final known sequence of 22nt containing sticky ends for the three main restriction enzymes in use (MluI, HpyCH4IV and AciI). This peculiar design allowed increasing the accuracy of clonal diversity estimation since the fixed-anchor sequence acts as a control for sequencing reliability in the barcode area. The theoretical number of different available barcodes per clone (412=16,777,216) far exceeds the requirements for not saturating the original diversity of the analyzed sample (on average composed by around 50.000 cells). We validated this novel approach by performing assays on serial dilutions of individual clones carrying known ISs. The precision rate obtained was averagely around 99.3%, while the worst error rate reaches at most the 1.86%, confirming the reliability of IS quantification. We successfully applied the barcoded-LC system to the analysis of clinical samples from a Wiskott Aldrich Syndrome GT patient, collecting to date 50,215 barcoded ISs from 94,052,785 sequencing reads

Autosomal Dominant Tubulointerstitial Kidney Disease with Adult Onset due to a Novel Renin Mutation Mapping in the Mature Protein

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a genetically heterogeneous renal disorder leading to progressive loss of renal function. ADTKD-REN is due to rare mutations in renin, all localized in the protein leader peptide and affecting its co-translational insertion in the endoplasmic reticulum (ER). Through exome sequencing in an adult-onset ADTKD family we identified a new renin variant, p.L381P, mapping in the mature protein. To assess its pathogenicity, we combined genetic data, computational and predictive analysis and functional studies. The L381P substitution affects an evolutionary conserved residue, co-segregates with renal disease, is not found in population databases and is predicted to be deleterious by in silico tools and by structural modelling. Expression of the L381P variant leads to its ER retention and induction of the Unfolded Protein Response in cell models and to defective pronephros development in zebrafish. Our work shows that REN mutations outside of renin leader peptide can cause ADTKD and delineates an adult form of ADTKD-REN, a condition which has usually its onset in childhood. This has implications for the molecular diagnosis and the estimated prevalence of the disease and points at ER homeostasis as a common pathway affected in ADTKD-REN, and possibly more generally in ADTKD

Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples

Background: Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. Methods: To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v.3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v.5 (Agilent Technologies). Results: Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values &gt;90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM\u2122 in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. Conclusions: High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants