Individual Exons Research Articles

Reduction of integrin alpha 4 activity through splice modulating antisense oligonucleotides

With recent approvals of antisense oligonucleotides as therapeutics, there is an increasing interest in expanding the application of these compounds to many other diseases. Our laboratory focuses on developing therapeutic splice modulating antisense oligonucleotides to treat diseases potentially amendable to intervention during pre-mRNA processing, and here we report the use of oligomers to down-regulate integrin alpha 4 protein levels. Over one hundred antisense oligonucleotides were designed to induce skipping of individual exons of the ITGA4 transcript and thereby reducing protein expression. Integrin alpha 4-mediated activities were evaluated in human dermal fibroblasts and Jurkat cells, an immortalised human T lymphocyte cell line. Peptide conjugated phosphorodiamidate morpholino antisense oligomers targeting ITGA4 were also assessed for their effect in delaying disease progression in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. With the promising results in ameliorating disease progression, we are optimistic that the candidate oligomer may also be applicable to many other diseases associated with integrin alpha 4 mediated inflammation. This highly specific strategy to down-regulate protein expression through interfering with normal exon selection during pre-mRNA processing should be applicable to many other gene targets that undergo splicing during expression.

SAT-048 A Map of Predicted Proteolytic Cleavage Products for the Soluble Human Protein Proteome: Intriguing Patterns

Protein hormones usually act via cell surface receptors linked to intracellular transduction pathways, they seem too complex and energetically expensive to be economical as single-message molecules. Proteolytic fragments of some protein hormones are known from earlier studies to have additional functions. Also, protein hormone translation sequences often contain multiple secreted products, e.g., poorly characterized pro-proteins or contraposed antagonistic hormones. If telescoped secondary functions are released during proteolytic processing at the synthetic cell, in circulation, or at target cells, organisms gain the efficiencies needed to use proteins as common signals. We have compiled a catalog of 2011 known soluble human protein hormone transcripts or transcript products and have now mapped them for their: predicted cleavages (PROSPER, Monash University) by 24 known proteases from 4 protease families (class: name and MEROPS number - Aspartate: HIV 1 retropepsin A02.001; Cysteine: cathepsin K C01.036, calpain 1 C02.001, caspase 1 C14.001, caspase 3 C14.003, caspase 7 C14.004, caspase 6 C14.005, caspase 8 C14.009; Metalloprotease: MMP 2 M10.003, MMP 9 M10.004, MMP 3 M10.005, MMP 7 M10.008; Serine: chymotrypsin A (bovine) S01.001, granzyme B (human) S01.010, elastase 2 S01.131, cathepsin G S01.133, granzyme B (mouse) S01.136, thrombin S01.217, plasmin S01.233, glutamyl peptidase I S01.269, furin S08.071, signal peptidase I S26.001, thylakoidal processing peptidase S26.008, signalase S26.010); the predicted secondary structures of the 887 unique transcripts; the known locations of the exon boundaries for the 459 canonical (Havanna annotation) transcripts; and the multiple alignment of the canonical transcripts. After exposure to all 24 proteases, 100% of the canonical transcripts still have left 8 +/- 7 (range 1 – 56) residual peptides of >10 amino acids long (M +/- SD, 18 +/- 8 residues; range, 10 – 67); only 1.19% of total fragments include single amino acid repeats of >4 residues. Although the cleavage prediction algorithm considers secondary structure, solvent access and surface charge as well as primary amino acid sequence, cleavage patterns are retained across multiple transcript isoforms and known bioactive transcript fragments down to peptides of 10-20 residues. Co-alignments of the proteolytic map, the secondary structure map, and the exon boundary map demonstrate a high propensity for overlap of these features including a 4.4-fold higher predicted proteolytic cleavage rate (% possible residues; p <0.01) within 3 residues of the exon boundaries versus sites >3 residues from the boundaries. The results suggest evolutionary retention of cleavage patterns allowing organismal access to secondary structures or functions, including nested secondary hormonal signals, encoded by single exons and obviating the need to preserve archaic individual exon genes.

Combined single-cell profiling of expression and DNA methylation reveals splicing regulation and heterogeneity

BackgroundAlternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic features, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remains poorly understood.ResultsWe applied parallel DNA methylation and transcriptome sequencing to differentiating human induced pluripotent stem cells to characterize splicing variation (exon skipping) and its determinants. Our results show that variation in single-cell splicing can be accurately predicted based on local sequence composition and genomic features. We observe moderate but consistent contributions from local DNA methylation profiles to splicing variation across cells. A combined model that is built based on genomic features as well as DNA methylation information accurately predicts different splicing modes of individual cassette exons. These categories include the conventional inclusion and exclusion patterns, but also more subtle modes of cell-to-cell variation in splicing. Finally, we identified and characterized associations between DNA methylation and splicing changes during cell differentiation.ConclusionsOur study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated link between DNA methylation variation and splicing.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Abstract 2467: Role of PCA3 in prostate cancer cell proliferation and progression

Abstract Background PCA3 is a long non-coding RNA used as an important biomarker in early prostate-cancer diagnosis but its biological functions are not understood. The aim of the study is to investigate the potential role of PCA3 in prostate cancer (PCa) initiation, growth and progression. Methods Human genomic DNA was used as templates for cloning of PCA3 gene. Primers were designed and polymerase chain reactions were performed. The 3 exons of PCA3 were cloned and confirmed by sequencing. To determine the impact of PCA3 and its individual exon in PCa cell growth and survival, we over-expressed them respectively in PC-3 and DU145 cell lines via transduction with lentiviral vector. qPCR was performed to examine the expression level of PCA3. To investigate the effects of PCA3 on prostate cancer cells, cell proliferation assay was performed by using trepan blue staining. Colony formation assays were performed to measure the clonogenic capacity of the cells after PCA3 overexpression and treatments with docetaxel. Results and conclusions Sequencing of PCA3 gene amplified from human tumor cells did not find mutations when compared to RefSeq. We overexpressed PCA3 in prostate cancer cells and found that increased PCA3 expression did not have significant impacts on DU145 or PC-3 cell proliferation. However, PCA3 exon 3 did increase the clonogenic capacity of DU145 cells when compared to the controls. DU145 cells with increased PCA3 exon 3 also presented increased resistance to docetaxel. No significance was observed in clonogenic capacity in transduced PC-3 cells, but PCA3 increased the motility of PC-3 cells. Our studies suggest that PCA3 can modulate prostate cancer cell clonogenic capacity, docetaxel resistance, and motility. Further studies are ongoing to determine the mechanisms involved. Citation Format: Bethanie Russel, Jiuhui Wang, Yuanqin Zhang, Daotai Nie. Role of PCA3 in prostate cancer cell proliferation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2467.

Framework for reanalysis of publicly available Affymetrix\xae GeneChip\xae data sets based on functional regions of interest

BackgroundSince the introduction of microarrays in 1995, researchers world-wide have used both commercial and custom-designed microarrays for understanding differential expression of transcribed genes. Public databases such as ArrayExpress and the Gene Expression Omnibus (GEO) have made millions of samples readily available. One main drawback to microarray data analysis involves the selection of probes to represent a specific transcript of interest, particularly in light of the fact that transcript-specific knowledge (notably alternative splicing) is dynamic in nature.ResultsWe therefore developed a framework for reannotating and reassigning probe groups for Affymetrix® GeneChip® technology based on functional regions of interest. This framework addresses three issues of Affymetrix® GeneChip® data analyses: removing nonspecific probes, updating probe target mapping based on the latest genome knowledge and grouping probes into gene, transcript and region-based (UTR, individual exon, CDS) probe sets. Updated gene and transcript probe sets provide more specific analysis results based on current genomic and transcriptomic knowledge. The framework selects unique probes, aligns them to gene annotations and generates a custom Chip Description File (CDF). The analysis reveals only 87% of the Affymetrix® GeneChip® HG-U133 Plus 2 probes uniquely align to the current hg38 human assembly without mismatches. We also tested new mappings on the publicly available data series using rat and human data from GSE48611 and GSE72551 obtained from GEO, and illustrate that functional grouping allows for the subtle detection of regions of interest likely to have phenotypical consequences.ConclusionThrough reanalysis of the publicly available data series GSE48611 and GSE72551, we profiled the contribution of UTR and CDS regions to the gene expression levels globally. The comparison between region and gene based results indicated that the detected expressed genes by gene-based and region-based CDFs show high consistency and regions based results allows us to detection of changes in transcript formation.

Whole genome sequencing reveals a novel deletion variant in the KIT gene in horses with white spotted coat colour phenotypes.

White spotting phenotypes in horses can range in severity from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for such phenotypes in horses. For the present study, we re-investigated a large horse family segregating a variable white spotting phenotype, for which conventional Sanger sequencing of the candidate genes' individual exons had failed to reveal the causative variant. We obtained whole genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~1.9-kb deletion spanning exons 10-13 of the KIT gene (chr3:77,740,239_77,742,136del1898insTATAT). In continuity with previously named equine KIT variants we propose to designate the newly identified deletion variant W22. We had access to 21 horses carrying the W22 allele. Four of them were compound heterozygous W20/W22 and had a completely white phenotype. Our data suggest that W22 represents a true null allele of the KIT gene, whereas the previously identified W20 leads to a partial loss of function. These findings will enable more precise genetic testing for depigmentation phenotypes in horses.

VCNet: vector-based gene co-expression network construction and its application to RNA-seq data

Building gene co-expression network (GCN) from gene expression data is an important field of bioinformatic research. Nowadays, RNA-seq data provides high dimensional information to quantify gene expressions in term of read counts for individual exons of genes. Such an increase in the dimension of expression data during the transition from microarray to RNA-seq era made many previous co-expression analysis algorithms based on simple univariate correlation no longer applicable. Recently, two vector-based methods, SpliceNet and RNASeqNet, have been proposed to build GCN. However, they failed to work when sample size is less than the number of exons. We develop an algorithm called VCNet to construct GCN from RNA-seq data to overcome this dimensional problem. VCNet performs a new statistical hypothesis test based on the correlation matrix of a gene-gene pair using the Frobenius norm. The asymptotic distribution of the new test is obtained under the null model. Simulation studies demonstrate that VCNet outperforms SpliceNet and RNASeqNet for detecting edges of GCN. We also apply VCNet to two expression datasets from TCGA database: the normal breast tissue and kidney tumour tissue, and the results show that the GCNs constructed by VCNet contain more biologically meaningful interactions than existing methods. VCNet is a useful tool to construct co-expression network. VCNet is open source and freely available from https://github.com/wangzengmiao/VCNet under GNU LGPL v3. dengmh@pku.edu.cn or nelsontang@cuhk.edu.hk. Supplementary data are available at Bioinformatics online.

Comparative Analysis and Classification of Cassette Exons and Constitutive Exons.

Alternative splicing (AS) is a major engine that drives proteome diversity in mammalian genomes and is a widespread cause of human hereditary diseases. More than 95% of genes in the human genome are alternatively spliced, and the most common type of AS is the cassette exon. Recent discoveries have demonstrated that the cassette exon plays an important role in genetic diseases. To discover the formation mechanism of cassette exon events, we statistically analyze cassette exons and find that cassette exon events are strongly influenced by individual exons that are smaller in size and that have a lower GC content, more codon terminations, and weaker splice sites. We propose an improved random-forest-based hybrid method of distinguishing cassette exons from constitutive exons. Our method achieves a high accuracy in classifying cassette exons and constitutive exons and is verified to outperform previous approaches. It is anticipated that this study will facilitate a better understanding of the underlying mechanisms in cassette exons.

Livebearing or egg-laying mammals: 27 decisive nucleotides of FAM168.

In the present study, we determine comprehensive molecular phylogenetic relationships of the novel myelin-associated neurite-outgrowth inhibitor (MANI) gene across the entire eukaryotic lineage. Combined computational genomic and proteomic sequence analyses revealed MANI as one of the two members of the novel family with sequence similarity 168 member (FAM168) genes, consisting of FAM168A and FAM168B, having distinct genetic differences that illustrate diversification in its biological function and genetic taxonomy across the phylogenetic tree. Phylogenetic analyses based on coding sequences of these FAM168 genes revealed that they are paralogs and that the earliest emergence of these genes occurred in jawed vertebrates such as Callorhinchus milii. Surprisingly, these two genes are absent in other chordates that have a notochord at some stage in their lives, such as branchiostoma and tunicates. In the context of phylogenetic relationships among eukaryotic species, our results demonstrate the presence of FAM168 orthologs in vertebrates ranging from Callorhinchus milii to Homo sapiens, displaying distinct taxonomic clusters, comprised of fish, amphibians, reptiles, birds, and mammals. Analyses of individual FAM168 exons in our sample provide new insights into the molecular relationships between FAM168A and FAM168B (MANI) on the one hand and livebearing and egg-laying mammals on the other hand, demonstrating that a distinctive intermediate exon 4, comprised of 27 nucleotides, appears suddenly only in FAM168A and there in the livebearing mammals only but is absent from all other species including the egg-laying mammals.

Molecular Cloning of Drebrin: Progress and Perspectives.

Chicken drebrin isoforms were first identified in the optic tectum of developing brain. Although the time course of protein expression was different in each drebrin isoform, the similarity between their protein structures was suggested by biochemical analysis of purified protein. To determine their protein structures, the cloning of drebrin cDNAs was conducted. Comparison between the cDNA sequences shows that all drebrin cDNAs are identical except that the internal insertion sequences are present or absent in their sequences. Chicken drebrin are now classified into three isoforms, namely, drebrins E1, E2, and A. Genomic cloning demonstrated that the three isoforms are generated by an alternative splicing of individual exons encoding the insertion sequences from single drebrin gene. The mechanism should be precisely regulated in cell-type-specific and developmental stage-specific fashion. Drebrin protein, which is well conserved in various vertebrate species, although mammalian drebrin has only two isoforms, namely, drebrin E and drebrin A, is different from chicken drebrin that has three isoforms. Drebrin belongs to an actin-depolymerizing factor homology (ADF-H) domain protein family. Besides the ADF-H domain, drebrin has other domains, including the actin-binding domain and Homer-binding motifs. Diversity of protein isoform and multiple domains of drebrin could interact differentially with the actin cytoskeleton and other intracellular proteins and regulate diverse cellular processes.

Genetic analysis of the PKHD1 gene with long-rang PCR sequencing.

PKHD1 gene mutations are found responsible for autosomal recessive polycystic kidney disease (ARPKD). However, it is inconvenient to detect the mutations by common polymerase chain reaction (PCR) because the open reading frame of PKHD1 is very long. Recently, long-range (LR) PCR is demonstrated to be a more sensitive mutation screening method for PKHD1 by directly sequencing. In this study, the entire PKHD1 coding region was amplified by 29 reactions to avoid the specific PCR amplification of individual exons, which generated the size of 1 to 7 kb products by LR PCR. This method was compared to the screening method with standard direct sequencing of each individual exon of the gene by a reference laboratory in 15 patients with ARPKD. The results showed that a total of 37 genetic changes were detected with LR PCR sequencing, which included 33 variations identified by the reference laboratory with standard direct sequencing. LR PCR sequencing had 100% sensitivity, 96% specificity, and 97.0% accuracy, which were higher than those with standard direct sequencing method. In conclusion, LR PCR sequencing is a reliable method with high sensitivity, specificity and accuracy for detecting genetic variations. It also has more intronic coverage and lower cost, and is an applicable clinical method for complex genetic analyses.

Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions

Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30–34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.

Age-dependent increase in Kalirin-9 and Kalirin-12 transcripts in human orbitofrontal cortex.

KALRN (KAL) is a Rho GEF that is highly involved in regulation of the actin cytoskeleton within dendrites. There are several isoforms of the protein that arise from differential splicing of KALRN's 66 exons. KAL isoforms have different functions in development. For example, overexpression of the KAL9 and KAL12 isoforms induce dendritic elongation in early development. However, in mature neurons KAL9 overexpression reduces dendritic length, a phenotype also observed in normal human ageing. We therefore hypothesized that KAL9 would have increased expression with age, and undertook to evaluate the expression of individual KALRN exons throughout the adult lifespan. Postmortem human brain grey matter from Brodmann's area (BA) 11 and BA47 derived from a cohort of 209 individuals without psychiatric or neurodegenerative disease, ranging in age from 16 to 91 years, were analysed for KALRN expression by Affymetrix exon array. Analysis of the exon array data in an isoform-specific manner, as well as confirmatory isoform-specific qPCR studies, indicated that the longer KAL9 and KAL12 isoforms demonstrated a statistically significant, but modest, increase with age. The small magnitude of the age effect suggests that inter-individual factors other than age likely contribute to a higher degree to KAL9 and KAL12 expression. In contrast to KAL9 and KAL12, global KALRN expression did not increase with age. Our work suggests that global measures of KALRN gene expression may be misleading and future studies should focus on isoform-specific quantification.

Multivariate eQTL mapping uncovers functional variation on the X-chromosome associated with complex disease traits.

Very few studies have investigated the associations between genetic polymorphisms and gene expression on the X-chromosome. This is a major bottleneck when conducting functional follow-up studies of trait-associated variants, as those identified in genome-wide association studies (GWAS). We used a multivariate approach to test the association between individual single nucleotide polymorphisms (SNPs) and exon expression levels measured in 356 Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from the Geuvadis RNA sequencing project to identify SNPs associated with variation in gene expression on the X-chromosome, which we refer to as eSNPs. At an FDR of 5%, we discovered 548 independent [linkage disequilibrium (LD) r (2)<0.1] eSNPs on the X-chromosome. Of these, 35 were in LD (r (2)>0.8) with previously published disease- or trait-associated variants identified through GWAS. One of the strongest eSNPs identified was rs35975601, which was associated with F8A1 expression (p value=3×10(-20)) and was in LD with a type 1 diabetes risk variant. Additionally, we identified a number of genes for which eSNPs were in LD with multiple diseases or traits, including DNASE1L1 which was mapped to bilirubin levels, type 1 diabetes and schizophrenia. Our results also indicate that multivariate exon-level analysis provides a more powerful approach than univariate gene-level analysis, particularly when SNPs influence the expression of different exons with different magnitude and/or direction of effect. The associations identified in our study may provide new insights into the molecular process by which gene expression may contribute to trait variation or disease risk in humans.

MUTATIONS OF THE SMARCB1 GENE IN HUMAN CANCERS

In the recent years, the full exome sequencing helped to reveal a set of mutations in the genes that are not oncogenes or tumor suppressor genes by definition, but play an important role in carcinogenesis and encode proteins involved in chromatin remodeling. Among chromatin remodeling systems, which operate through the ATP-dependent mechanism, the complex SWI/ SNF attracts the great attention. The complex consists of the catalytic ATPase (SMARCA2/4), a group of conservative core subunits (SMARCB1, SMARCC1/2), and variant subunits. Abnormalities in the genes coding for each of these components have been identified as driver mutations in various human tumors. The SMARCB1 gene is of interest for practical oncogenetics, with its typical genotype-phenotype correlations. Germinal inactivating mutations (frameshift insertions/deletions, full deletions of the gene, nonsense mutations) lead to development of rhabdoid tumors in the kidneys and the brain in children in their first years of life, or even in utero. These tumors are highly malignant (Rhabdoid Tumor Predisposition Syndrome 1 – RTPS1). If a mutation carrier survives his/hers four years of life without manifestation RTPS1 with a missense mutation or has the mutation in the hot spot of the first or the last exon, then he/she will not develop rhabdoid tumors, but after 20 years of life, shwannomatosis may develop as multiple benign tumors of peripheral nerves. Finally, some point mutations in the exons 8–9 can result in Coffin-Siris syndrome characterized by mental retardation and developmental disorders, but no neoplasms. In this regard, rational referral of patients for direct DNA diagnostics of each of the described disease entities plays an important role, based on respective minimal criteria, as well as necessity of further development of NGS technologies (full genome and full exome sequencing) that are able to sequence not only individual exons, but all candidate genes of the disorders.

ExSurv: A Web Resource for Prognostic Analyses of Exons Across Human Cancers Using Clinical Transcriptomes.

Survival analysis in biomedical sciences is generally performed by correlating the levels of cellular components with patients’ clinical features as a common practice in prognostic biomarker discovery. While the common and primary focus of such analysis in cancer genomics so far has been to identify the potential prognostic genes, alternative splicing – a posttranscriptional regulatory mechanism that affects the functional form of a protein due to inclusion or exclusion of individual exons giving rise to alternative protein products, has increasingly gained attention due to the prevalence of splicing aberrations in cancer transcriptomes. Hence, uncovering the potential prognostic exons can not only help in rationally designing exon-specific therapeutics but also increase specificity toward more personalized treatment options. To address this gap and to provide a platform for rational identification of prognostic exons from cancer transcriptomes, we developed ExSurv (https://exsurv.soic.iupui.edu), a web-based platform for predicting the survival contribution of all annotated exons in the human genome using RNA sequencing-based expression profiles for cancer samples from four cancer types available from The Cancer Genome Atlas. ExSurv enables users to search for a gene of interest and shows survival probabilities for all the exons associated with a gene and found to be significant at the chosen threshold. ExSurv also includes raw expression values across the cancer cohort as well as the survival plots for prognostic exons. Our analysis of the resulting prognostic exons across four cancer types revealed that most of the survival-associated exons are unique to a cancer type with few processes such as cell adhesion, carboxylic, fatty acid metabolism, and regulation of T-cell signaling common across cancer types, possibly suggesting significant differences in the posttranscriptional regulatory pathways contributing to prognosis.

The clustered protocadherin endolysosomal trafficking motif mediates cytoplasmic association

BackgroundClustered protocadherins (Pcdhs) are a large family of neural cadherin-like proteins encoded by individual exons located within three gene clusters. Each exon codes an extracellular, transmembrane, and proximal cytoplasmic domain. These “variable” regions may be spliced to a constant cytoplasmic moiety encoded at the end of a cluster. Pcdh extracellular domains mediate homophilic cell-cell binding but their cytoplasmic domains cause intracellular retention and may negatively regulate Pcdh cell-cell binding. Pcdhs can be found at the cell surface in neurons and other cells but are also, unlike classical cadherins, prominently trafficked to the endolysosome system. It was previously found that a segment within the variable portion of the Pcdh-γA3 cytoplasmic domain (VCD) was shown to be necessary for endolysosomal trafficking.ResultsHere it is shown that this same VCD segment can mediate cytoplasmic association among Pcdhs from the different clusters. Internal deletions within this VCD region (termed here the VCD motif) that disrupt the association altered trafficking of Pcdh-γA3 in the endolysosomal system while deletions outside VCD motif did not affect trafficking.ConclusionsThe results show that Pcdhs associate cytoplasmically via a motif within the VCD and that this is critical for Pcdh trafficking. Given that truncation at the VCD motif alters endolysosomal trafficking of Pcdhs, the VCD interaction described here may provide new insights into the dynamic nature of Pcdh mediated cell-cell interactions.

Full-length HLA-DRB1 coding sequences generated by a hemizygous RNA-SBT approach.

Currently 1582 HLA-DRB1 alleles have been identified in the IMGT/HLA database (v3.18). Among those alleles, more than 90% have incomplete allele sequences, which complicates the analysis of the functional relevance of polymorphism beyond exon 2. The polymorphic index of each individual exon of the currently known allele sequences, shows that polymorphism is present in all exons, albeit not equally abundant. Full-length HLA-DRB1 RNA sequencing identifies polymorphism of the complete coding region. Here we describe a hemizygous full-length RNA sequence-based typing (SBT) approach based on group-specific HLA-DRB1 amplification and subsequent sequencing. RNA full-length sequences can easily be accessed because of the short amplicon length (801 bp). The RNA-SBT approach was successfully validated on a panel of DRB1 alleles having fully known coding sequences according to the IMGT/HLA database, and cover all serological equivalents. Subsequently, the approach was applied on a panel of 54 alleles with incomplete allele sequences, resulting in full-length coding sequences and the identification of one new and one corrected allele. This study shows the universal applicability of the RNA-based sequencing approach to identify full-length coding sequences and to define the polymorphic content of HLA-DRB1 alleles.

Abstract 4884: Highly sensitive fusion transcript detection and quantification in cancer

Abstract Gene fusion detection in cancer samples can provide tumor-specific information for cancer research, clinical diagnosis and targeted treatment. Common fusion detection methods such as qPCR and FISH are restricted to known fusion junctions and limited in the number of genes that can be detected in parallel. In contrast, RNA sequencing is a powerful approach for simultaneous discovery of all possible fusion junctions in a single reaction. But, the sequencing depth required for sensitive detection of fusions from whole-transcriptome libraries can be cost-prohibitive. Here we describe a cancer-specific capture-based approach for fusion detection by RNA sequencing that requires only a fraction of the sequencing depth of whole-transcriptome methods. We designed oligo probes that densely target coding regions of over 200 clinically relevant gene fusions and cancer-associated genes. This oligo panel was used to capture cancer-specific fusions from total RNA-Seq libraries. We used commonly studied cancer cell lines including MCF-7, K562, PC-3, LnCAP, A431 and Universal Human Reference RNA (UHRR) to compare the sensitivity of fusion detection across three RNA-Seq library prep methods: (1) cancer panel library capture (2) whole-transcriptome library capture and (3) PolyA selection. We show that probes targeting individual exons can robustly capture well-characterized cancer gene fusions such as BCR-ABL and BCAS4-BCAS3, as well as translocations where fusion junctions are unknown. Furthermore, these comparisons demonstrate the enhanced sequencing efficiency of the targeted cancer panel, while maintaining highly accurate quantitation of gene expression. We show that selective enrichment of RNA-Seq libraries with cancer-specific capture probes enables high-resolution mapping of genomic rearrangements in patient cancer samples, even those derived from FFPE, facilitating sequencing studies that were not previously possible. Citation Format: Lisa C. Watson, Stephen M. Gross, Irina Khrevtukova, Smita Pathak, Claire Attwooll, Jason Goode, Anthony Mai, Gary P. Schroth. Highly sensitive fusion transcript detection and quantification in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4884. doi:10.1158/1538-7445.AM2015-4884