Exonic Splicing Enhancer Research Articles

Curated multiple sequence alignment for the Adenomatous Polyposis Coli (APC) gene and accuracy of in silico pathogenicity predictions.

Computational algorithms are often used to assess pathogenicity of Variants of Uncertain Significance (VUS) that are found in disease-associated genes. Most computational methods include analysis of protein multiple sequence alignments (PMSA), assessing interspecies variation. Careful validation of PMSA-based methods has been done for relatively few genes, partially because creation of curated PMSAs is labor-intensive. We assessed how PMSA-based computational tools predict the effects of the missense changes in the APC gene, in which pathogenic variants cause Familial Adenomatous Polyposis. Most Pathogenic or Likely Pathogenic APC variants are protein-truncating changes. However, public databases now contain thousands of variants reported as missense. We created a curated APC PMSA that contained >3 substitutions/site, which is large enough for statistically robust in silico analysis. The creation of the PMSA was not easily automated, requiring significant querying and computational analysis of protein and genome sequences. Of 1924 missense APC variants in the NCBI ClinVar database, 1800 (93.5%) are reported as VUS. All but two missense variants listed as P/LP occur at canonical splice or Exonic Splice Enhancer sites. Pathogenicity predictions by five computational tools (Align-GVGD, SIFT, PolyPhen2, MAPP, REVEL) differed widely in their predictions of Pathogenic/Likely Pathogenic (range 17.5-75.0%) and Benign/Likely Benign (range 25.0-82.5%) for APC missense variants in ClinVar. When applied to 21 missense variants reported in ClinVar and securely classified as Benign, the five methods ranged in accuracy from 76.2-100%. Computational PMSA-based methods can be an excellent classifier for variants of some hereditary cancer genes. However, there may be characteristics of the APC gene and protein that confound the results of in silico algorithms. A systematic study of these features could greatly improve the automation of alignment-based techniques and the use of predictive algorithms in hereditary cancer genes.

Synonymous Mutation in DKC1 Causes Telomerase RNA Insufficiency Manifesting as Familial Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common of short telomere phenotypes. Familial clustering of IPF is common, but the genetic basis remains unknown in more than one-half of cases. We identified a 65-year-old man with familial IPF, short telomere length, and low telomerase RNA levels. He was diagnosed with a short telomere syndrome after developing hematologic complications post-lung transplantation, but no mutations were identified in a clinical testing pipeline. What is the molecular basis underlying the familial IPF and low telomerase RNA levels in this patient? We analyzed whole-genome sequence data and performed functional molecular studies on cells derived from the patient and his family. We identified a previously unreported synonymous variant c.942G>A p.K314K in DKC1, the gene encoding the dyskerin ribonucleoprotein, which is required for telomerase RNA biogenesis. The mutation created a competing de novo exonic splicing enhancer, and the misspliced product was degraded by nonsense-mediated decay causing an overall dyskerin deficiency in mutation carriers. In silico tools identified other rare silent DKC1 variants that warrant functional evaluation if found in patients with short telomere-mediated disease. Our data point to silent mutation in telomere maintenance genes as a mechanism of familial pulmonary fibrosis. In contrast to DKC1 missense mutations, which primarily manifest in children as dyskeratosis congenita, hypomorphic mutations affecting dyskerin levels likely have a predilection to presenting in adults as pulmonary fibrosis.

Novel loss-of-function mutations in COCH cause autosomal recessive nonsyndromic hearing loss.

COCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly hearing loss in mice and humans. Two forms of hearing loss are linked to mutations in COCH, the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing, and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense, and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used minigene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA splicing, and highlights the need to investigate the effect of coding variants on RNA splicing.

Mutational bias and the protein code shape the evolution of splicing enhancers

Exonic splicing enhancers (ESEs) are enriched in exons relative to introns and bind splicing activators. This study considers a fundamental question of co-evolution: How did ESE motifs become enriched in exons prior to the evolution of ESE recognition? We hypothesize that the high exon to intron motif ratios necessary for ESE function were created by mutational bias coupled with purifying selection on the protein code. These two forces retain certain coding motifs in exons while passively depleting them from introns. Through the use of simulations, genomic analyses, and high throughput splicing assays, we confirm the key predictions of this hypothesis, including an overlap between protein and splicing information in ESEs. We discuss the implications of mutational bias as an evolutionary driver in other cis-regulatory systems.

Structural disruption of exonic stem-loops immediately upstream of the intron regulates mammalian splicing.

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem–loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

Shani mutation in mouse affects splicing of Spata22 and leads to impaired meiotic recombination.

Recombination is crucial for chromosome pairing and segregation during meiosis. SPATA22, along with its direct binding partner and functional collaborator, MEIOB, is essential for the proper repair of double-strand breaks (DSBs) during meiotic recombination. Here, we describe a novel point-mutated allele (shani) of mouse Spata22 that we isolated in a forward genetic screen. shani mutant mice phenocopy Spata22-null and Meiob-null mice: mutant cells appear to form DSBs and initiate meiotic recombination, but are unable to complete DSB repair, leading to meiotic prophase arrest, apoptosis and sterility. shani mutants show precocious loss of DMC1 foci and improper accumulation of BLM-positive recombination foci, reinforcing the requirement of SPATA22-MEIOB for the proper progression of meiotic recombination events. The shani mutation lies within a Spata22 coding exon and molecular characterization shows that it leads to incorrect splicing of the Spata22 mRNA, ultimately resulting in no detectable SPATA22 protein. We propose that the shani mutation alters an exonic splicing enhancer element (ESE) within the Spata22 transcript. The affected DNA nucleotide is conserved in most tetrapods examined, suggesting that the splicing regulation we describe here may be a conserved feature of Spata22 regulation.

SUN-723 CDH2 Gene Analysis in a Cohort of Patients with Congenital Hypopituitarism

Introduction: Hypopituitarism is defined as a deficiency of one or more pituitary hormones. Pathogenic allelic variants in genes implicated in pituitary development were associated in 15% of the patients with congenital hypopituitarism (CH). To improve the molecular diagnosis we performed whole exome sequencing of ten patients born from consanguineous parents with CH. One patient with GH, TSH, ACTH and LH/FSH deficiencies presented an allelic variant c.865G>A, p.V289I in CDH2 gene (exon 7) in homozygous state that was absent in populational databanks. CDH2 produces an N-cadherin protein implicated in cellular adhesion and is responsible for epithelial-mesenchymal transition during pituitary development and differentiation. Aim: To analyze the CDH2 gene in a cohort of unrelated patients with CH. Methods: We selected 143 patients with CH from a single Brazilian center. Genomic DNA, extracted by salting out technique, was submitted to PCR amplification of 15 coding regions, except CG rich exon 1, of the CDH2 gene followed by the Sanger sequencing. Rare allelic variant frequency (MAF<1%) was searched in the populational data bank (ExAC, gnomAD, ABraom). Bioinformatic sites (Human Splicing Finder, Polyphen2, Mutation Taster and Mutation assessor) were used to look for deleterious effects. Results: Three allelic variants were found in this cohort. The allelic variant CDH2 (c.865G>A, p.V289I) was found in heterozygous state in a male patient with short stature diagnosed with GH and TSH deficiencies at the age of 11 that evolved with LH/FSH and ACTH deficiencies. Family segregation showed 3 among 11 normal siblings heterozygous carriers. This variant is rare, in heterozygous state, in populational data bank and it was predicted as deleterious or possibly harmful. The allelic variant c.1202C> A (p.A401D), in exon 9, was found in heterozygous state in a female patient with isolated GH deficiency and intellectual disability. The variant was absent in the databases and predicted as deleterious or disease-causing. The variant was absent in the mother and stepsister and the father was not available for testing. The c.1430_1431delCCinsTG allelic variant (p.P477L) was found in heterozygous state in a patient with septo-optic dysplasia, GH, TSH and ACTH deficiencies. It was absent in the databases and was predicted as deleterious or disease causing. The Human Splicing Finder predicted exonic splicing enhancer breakdown leading to the loss of 93 nucleotides. Normal mother is heterozygous carrier suggesting incomplete penetrance. Conclusion: Heterozygous variants in CDH2 were found in 2% of a cohort of Brazilian patients with congenital hypopituitarism and none in homozygous or compound heterozygous state. Further CDH2 analyses in unrelated patients from different ethnic backgrounds are needed to establish the role CDH2 variants in the etiology of congenital hypopituitarism.

Design and Testing of RNA Therapeutics to Block VEGFR2 and EGFR Expression in Human Glioblastoma

Glioblastoma multiforme (GBM), is the most common and aggressive malignant primary brain tumor with a median survival of 15 months with standard care. Current therapies are limited by the blood brain barrier. Tumor blood vessel formation depends on vascular endothelial growth factor receptor 2 (VEGFR2), while tumor cell proliferation is stimulated by epidermal growth factor receptor (EGFR). Both are important for tumor cell survival. Our novel approach delivers directly to the tumor cells, genes encoding RNA therapeutics. Our strategy is to alter the splicing mechanism of the VEGFR2 and EGFR genes to block their activation, thus stop tumor cell angiogenesis and growth Our current RNA therapeutics demonstrate significant reductions in EGFR and VEGFR2 expression. To increase the efficacy of our RNA therapeutics, we are fine‐tune targeting regions of EGFR and VEGFR2, as well as recruiting splicing silencers and enhancers to the pre‐mRNA transcripts. Can we generate therapy vectors that encode antisense RNA therapeutics that either block or activate splicing motifs and alter the expression of functional VEGFR2 and EGFR in GBM significantly reducing tumor growth? Forty‐five antisense sequences were designed to target the EGFR gene and nine antisense sequences for VEGFR2, to potentially block their activation. The antisense sequences were cloned into our pAAV‐U7‐smOPT therapy vector. In addition, multiple strategies were used to clone the exonic splicing silencer 4G‐quadruplex and five distinct exonic splicing enhancer motifs into the RNA anti‐sense therapy vector, which recruit serine‐arginine (SR) proteins, directing the spliceosome to a desired splice site or intronic poly‐adenylation site. Moreover, another aspect of this research is to isolate the mRNA of multiple tyrosine kinase receptors from GBM cancer cells, clone the cDNA into a T7 expression vector to transcribe control RNA to use in our high throughput sequencing experiments. Multiple GBM cell lines, including U87 and SKMG3 were transfected with our novel therapies. Total mRNA was collected, analyzed, and compared to the same cell lines without treatment, showing a significant reduction in target expression. The collected data allow analysis to measure efficacy of current anti‐VEGFR2 and anti‐EGFR therapeutic strategies which will be tested in a mouse model using adeno‐associated virus (AAV) vector.

Association of Gene Variations in Ionotropic Glutamate Receptor and Attention-Deficit/Hyperactivity Disorder in the Chinese Population: A Two-Stage Case–Control Study

Objective: The aim of this study was to comprehensively explore the relationship between genetic variations within GRIN2A, GRIN2B, GRIK1, GRIK4, GRID2, and ADHD. Method: Genotyping was performed with the Sequenom MassARRAY system in a two-stage case–control study. ADHD symptoms were assessed using the Swanson, Nolan, and Pelham version IV scale and the Integrated Visual and Auditory Continuous Performance Test. In silico analysis was performed with website resources. Results: GRID2 rs1385405 showed a significant association with ADHD risk in the codominant model (OR = 2.208, 95% CI = [1.387, 3.515]) in the first stage and in the codominant model (OR = 1.874, 95% CI = [1.225, 2.869]) and recessive model (OR = 1.906, 95% CI = [1.265, 2.873]) in the second stage and related to inattention and hyperactivity symptom. In addition, rs1385405 disturbed the activity of exonic splicing enhancer and mediated GRID2 gene expression in the frontal cortex. Conclusion: our data provided evidence for the participation of GRID2 variants in conferring the risk of ADHD.

Detection of polymorphism within leptin gene in Egyptian river buffalo and predict its effects on different molecular levels

BackgroundLeptin (LEP) regulates the glucose homeostasis directly and centrally by the regulation of the insulin levels or indirectly by alternation of the levels of the other glucose metabolism regulator hormones. The present investigation studied the polymorphism in LEP gene which is related to fertility in 81 female Egyptian river buffalo. ResultsThe PCR-RFLP pattern of the gene using the restriction enzyme Eco91I showed that all the animals had monomorphic pattern in the studied gene which consists of CC. A 511-bp fragment from LEP gene was amplified and sequenced. The homology between the amplified LEP gene fragment in buffalo and cattle, sheep, goat, human, and mouse on the nucleotides sequence level was 99, 97, 97, 87, and 79%, respectively, and on the translated amino acids sequence level was 100, 98, 98, 85, and 82%, respectively. Several SNPs were detected; among them, the T27C SNP disrupted an intronic splicing silencer. The A114G, A310G, G263A, and G379A SNPs disrupt exonic splicing enhancers, and the last two SNPs create new exonic splicing enhancers. The A114G, C163A, A211G, G288A, A310G, A322G, G330C, C348T, T360C, and G379A SNPs cause S71G, T87 N, N103S, E129K, E136G, Y140C, E143Q, R149W, S153P, and R159Q amino acids mutations. N103S, E129K, E136G, Y140C, E143Q, and S153P were classified as deleterious mutations. Y140, E143, N103, and R149 were the most conserved among the mutated amino acids. S71G only increased the stability of the leptin protein while the remaining mutations decreased it. ConclusionFour SNPs were revealed among the tested animals. Twenty-one SNPs were found between the sequenced amplicon and the buffalo records in the Genbank. Some SNPs were predicted to have several effects on different biological processes like mRNA splicing, protein stability, and the gene functions.

SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway.

MDM2 is an oncogene and critical negative regulator of tumor suppressor p53. Genotoxic stress causes alternative splicing of MDM2 transcripts, which leads to alterations in p53 activity and contributes to tumorigenesis. MDM2-ALT1 is one of the alternatively spliced transcripts predominantly produced in response to genotoxic stress, and is comprised of terminal coding exons 3 and 12. Previously, we found that SRSF1 induces MDM2-ALT1 by promoting MDM2 exon 11 skipping. Here we report that splicing regulator SRSF2 antagonizes the regulation of SRSF1 by facilitating the inclusion of exon 11 through binding at two conserved exonic splicing enhancers. Overexpression of SRSF2 reduced the generation of MDM2-ALT1 under genotoxic stress, whereas SRSF2 knockdown induced the expression of MDM2-ALT1 in the absence of genotoxic stress. Blocking the exon 11 SRSF2-binding sites using oligonucleotides promoted MDM2-ALT1 splicing and induced p53 protein expression, and apoptosis in p53 wild-type cells. The regulation of MDM2 splicing by SRSF2 is also conserved in mice, as mutation of one SRSF2-binding site in Mdm2 exon 11, using CRISPR-Cas9, increased the expression of the MDM2-ALT1 homolog Mdm2-MS2. IMPLICATIONS: Taken together, the data indicate that modulating MDM2 splicing may be a useful tool for fine-tuning p53 activity in response to genotoxic stress.

Analysis of FUS, PFN2, TDP-43, and PLS3 as potential disease severity modifiers in spinal muscular atrophy.

ObjectiveTo investigate mutations in genes that are potential modifiers of spinal muscular atrophy (SMA) severity.MethodsWe performed a hypothesis-based search into the presence of variants in fused in sarcoma (FUS), transactive response DNA-binding protein 43 (TDP-43), plastin 3 (PLS3), and profilin 2 (PFN2) in a cohort of 153 patients with SMA types 1–4, including 19 families. Variants were detected with targeted next-generation sequencing and confirmed with Sanger sequencing. Functional effects of the identified variants were analyzed in silico and for PLS3, by analyzing expression levels in peripheral blood.ResultsWe identified 2 exonic variants in FUS exons 5 and 6 (p.R216C and p.S135N) in 2 unrelated patients, but clinical effects were not evident. We identified 8 intronic variants in PLS3 in 33 patients. Five PLS3 variants (c.1511+82T>C; c.748+130 G>A; c.367+182C>T; c.891-25T>C (rs145269469); c.1355+17A>G (rs150802596)) potentially alter exonic splice silencer or exonic splice enhancer sites. The variant c.367+182C>T, but not RNA expression levels, corresponded with a more severe phenotype in 1 family. However, this variant or level of PLS3 expression did not consistently correspond with a milder or more severe phenotype in other families or the overall cohort. We found 3 heterozygous, intronic variants in PFN2 and TDP-43 with no correlation with clinical phenotype or effects on splicing.ConclusionsPLS3 and FUS sequence variants do not modify SMA severity at the population level. Specific variants in individual patients or families do not consistently correlate with disease severity.

A Depletion of Stop Codons in lincRNA is Owing to Transfer of Selective Constraint from Coding Sequences.

Although the constraints on a gene’s sequence are often assumed to reflect the functioning of that gene, here we propose transfer selection, a constraint operating on one class of genes transferred to another, mediated by shared binding factors. We show that such transfer can explain an otherwise paradoxical depletion of stop codons in long intergenic noncoding RNAs (lincRNAs). Serine/arginine-rich proteins direct the splicing machinery by binding exonic splice enhancers (ESEs) in immature mRNA. As coding exons cannot contain stop codons in one reading frame, stop codons should be rare within ESEs. We confirm that the stop codon density (SCD) in ESE motifs is low, even accounting for nucleotide biases. Given that serine/arginine-rich proteins binding ESEs also facilitate lincRNA splicing, a low SCD could transfer to lincRNAs. As predicted, multiexon lincRNA exons are depleted in stop codons, a result not explained by open reading frame (ORF) contamination. Consistent with transfer selection, stop codon depletion in lincRNAs is most acute in exonic regions with the highest ESE density, disappears when ESEs are masked, is consistent with stop codon usage skews in ESEs, and is diminished in both single-exon lincRNAs and introns. Owing to low SCD, the maximum lengths of pseudo-ORFs frequently exceed null expectations. This has implications for ORF annotation and the evolution of de novo protein-coding genes from lincRNAs. We conclude that not all constraints operating on genes need be explained by the functioning of the gene but may instead be transferred owing to shared binding factors.

Characterization of splice-altering mutations in inherited predisposition to cancer

Mutations responsible for inherited disease may act by disrupting normal transcriptional splicing. Such mutations can be difficult to detect, and their effects difficult to characterize, because many lie deep within exons or introns where they may alter splice enhancers or silencers or introduce new splice acceptors or donors. Multiple mutation-specific and genome-wide approaches have been developed to evaluate these classes of mutations. We introduce a complementary experimental approach, cBROCA, which yields qualitative and quantitative assessments of the effects of genomic mutations on transcriptional splicing of tumor suppressor genes. cBROCA analysis is undertaken by deriving complementary DNA (cDNA) from puromycin-treated patient lymphoblasts, hybridizing the cDNA to the BROCA panel of tumor suppressor genes, and then multiplex sequencing to very high coverage. At each splice junction suggested by split sequencing reads, read depths of test and control samples are compared. Significant Z scores indicate altered transcripts, over and above naturally occurring minor transcripts, and comparisons of read depths indicate relative abundances of mutant and normal transcripts. BROCA analysis of genomic DNA suggested 120 rare mutations from 150 families with cancers of the breast, ovary, uterus, or colon, in >600 informative genotyped relatives. cBROCA analysis of their transcripts revealed a wide variety of consequences of abnormal splicing in tumor suppressor genes, including whole or partial exon skipping, exonification of intronic sequence, loss or gain of exonic and intronic splicing enhancers and silencers, complete intron retention, hypomorphic alleles, and combinations of these alterations. Combined with pedigree analysis, cBROCA sequencing contributes to understanding the clinical consequences of rare inherited mutations.

A single-nucleotide polymorphism induced alternative splicing in Tacr3 involves in hypoxic-ischemic brain damage

Single-nucleotide polymorphism (SNP) and Alternative splicing (AS) were found to be implicated in certain diseases, nevertheless, the contributions of mRNA SNPs and AS to pathogenesis in developing rat brains with hypoxic-ischemic encephalopathy (HIE) remained largely vague. Additionally, the disease associated with Tacr3 was normosmic congenital hypogonadotropic hypogonadism, while the relationship between HIE and Tacr3 remained largely elusive. The current study was designed to investigate the differentially expressed mRNAs and related SNPs as well as AS in neonatal rats subjected to HIE to identify if the exhibition of AS was associated with SNPs under pathological condition. Firstly, we used postnatal day 7 Sprague-Dawley rats to construct neonatal HIE model, and analyzed the expression profiles of SNP mRNA in hypoxic-ischemic (HI) and sham brains by using RNA sequencing. Then four genes, including Mdfic, Lpp, Bag3 and Tacr3, connecting with HIE and exhibiting SNPs and AS were identified by bioinformatics analysis. Moreover, combined with exonic splicing enhancer (ESE) and alternative splice site predictor (ASSP) analysis, we found that Tacr3 is associated specifically with HIE through 258547789 G > A SNP in inside the Alt First Exon and 258548573 G > A SNP in outside the Alt First Exon. Taken together, our study provides new evidence to understand the role of Tacr3 in HIE and it is possibly a potential target for the treatment of HIE in future clinic trial.

The functional mechanisms of mutations in myelodysplastic syndrome

Overlapping spectrum of mutated genes affected in myelodysplastic syndrome (MDS) and primary acute myeloid leukemia suggest common pathogenic mechanisms. However, the frequencies of specific mutations are significantly different between them, which implies they might determine specific disease phenotype. For instance, there are overrepresentations of mutations in RNA splicing factors or epigenetic regulators in MDS. We provide an overview of recent advances in our understanding of the biology of MDS and related disorders. Our focus is how mutations of in splicing factors or epigenetic regulators identified in MDS patients demonstrate phenotypes in knockin/knockout mouse models. For instance, mutant Srsf2 mice could alter Srsf2's normal sequence-specific RNA binding activity. It exhibited changing in the recognition of specific exonic splicing enhancer motifs to drive recurrent missplicing of Ezh2, which reduces Ezh2 expression by promoting nonsense-mediated decay. Consistent with this, SRSF2 mutations are mutually exclusive with EZH2 loss-of-function mutations in MDS patients. We also review how gene editing technology identified unique associations between pathogenic mechanisms and targeted therapy using lenalidomide, including: (i) how haploinsufficiency of the genes located in the commonly deleted region in del(5q) MDS patients promotes MDS; (ii) how lenalidomide causes selective elimination of del(5q) MDS cells; and (iii) why del(5q) MDS patients become resistant to lenalidomide. Thus, this review describes our current understanding of the mechanistic and biological effects of mutations in spliceosome and epigenetic regulators by comparing wild-type normal to mutant function as well as a brief overview of the recent progresses in MDS biology.

Massive computational identification of somatic variants in exonic splicing enhancers using The Cancer Genome Atlas.

Owing to the development of next‐generation sequencing (NGS) technologies, a large number of somatic variants have been identified in various types of cancer. However, the functional significance of most somatic variants remains unknown. Somatic variants that occur in exonic splicing enhancer (ESE) regions are thought to prevent serine and arginine‐rich (SR) proteins from binding to ESE sequence motifs, which leads to exon skipping. We computationally identified somatic variants in ESEs by compiling numerous open‐access datasets from The Cancer Genome Atlas (TCGA). Using somatic variants and RNA‐seq data from 9635 patients across 32 TCGA projects, we identified 646 ESE‐disrupting variants. The false positive rate of our method, estimated using a permutation test, was approximately 1%. Of these ESE‐disrupting variants, approximately 71% were located in the binding motifs of four classical SR proteins. ESE‐disrupting variants occurred in proportion to the number of somatic variants, but not necessarily in the specific genes associated with the biological processes of cancer. Existing bioinformatics tools could not predict the pathogenicity of ESE‐disrupting variants identified in this study, although these variants could cause exon skipping. We demonstrated that ESE‐disrupting nonsense variants tended to escape nonsense‐mediated decay surveillance. Using integrated analyses of open access data, we could specifically identify ESE‐disrupting variants. We have generated a powerful tool, which can handle datasets without normal samples or raw data, and thus contribute to reducing variants of uncertain significance because our statistical approach only uses the exon‐junction read counts from the tumor samples.

SRSF3-Regulated RNA Alternative Splicing Promotes Glioblastoma Tumorigenicity by Affecting Multiple Cellular Processes.

Misregulated alternative RNA splicing (AS) contributes to the tumorigenesis and progression of human cancers, including glioblastoma (GBM). Here, we showed that a major splicing factor, serine and arginine rich splicing factor 3 (SRSF3), was frequently upregulated in clinical glioma specimens and that elevated SRSF3 was associated with tumor progression and a poor prognosis for patients with glioma. In patient-derived glioma stem-like cells (GSC), SRSF3 expression promoted cell proliferation, self-renewal, and tumorigenesis. Transcriptomic profiling identified more than 1,000 SRSF3-affected AS events, with a preference for exon skipping in genes involved with cell mitosis. Motif analysis identified the sequence of CA(G/C/A)CC(C/A) as a potential exonic splicing enhancer for these SRSF3-regulated exons. To evaluate the biological impact of SRSF3-affected AS events, four candidates were selected whose AS correlated with SRSF3 expression in glioma tissues, and their splicing pattern was modified using a CRISPR/Cas9 approach. Two functionally validated AS candidates were further investigated for the mechanisms underlying their isoform-specific functions. Specifically, following knockout of SRSF3, transcription factor ETS variant 1 (ETV1) gene showed exon skipping at exon 7, while nudE neurodevelopment protein 1 (NDE1) gene showed replacement of terminal exon 9 with a mutually exclusive exon 9'. SRSF3-regulated AS of these two genes markedly increased their oncogenic activity in GSCs. Taken together, our data demonstrate that SRSF3 is a key regulator of AS in GBM and that understanding mechanisms of misregulated AS could provide critical insights for developing effective therapeutic strategies against GBMs. SIGNIFICANCE: SRSF3 is a significant regulator of glioma-associated alternative splicing, implicating SRSF3 as an oncogenic factor that contributes to the tumor biology of GBM.

CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation

The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.

Abstract 776: MYBPC3 Mutations in Indian Patients With Hypertrophic Cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is characterized by thickening of the ventricular wall, myocyte disarray, and fibrosis, resulting in impaired cardiac function and leading to adverse ventricular remodeling, heart failure and increased risk for sudden cardiac death. Importantly, mutations in the gene encoding cardiac myosin binding protein-C ( MYBPC3 ), a muscle contractile protein,account for approximately 50% of the reported HCM cases. However, the prevalence of penetrance of MYBPC3 mutations in Indian patients with HCM is yet to be systematically studied. Methods: The Institutional Human Ethics Committee at the University of Puneapproved the present study. HCM patients undergoing treatment in theCardiology Unit of Bharti Hospital, Pune, were identified. With the approval of written informed consent, blood samples were obtained from all patients and their relatives and screened by single-strand conformation polymorphism (SSCP) and direct DNA sequencing, followed by correlation with clinical outcome. Results: Of the 23 probands screened in this study, one, #16, showed a mutation in exon 07 (A/G) at chromosomal position 47370041, causing a protein change at position NP_000247.2:p.S236G, and reported as SNP status ID rs386584787. The mutation was localizedto the immunoglobulin domain of cardiac myosin binding protein-C, causing an amino acid change from serine to glycine with a score of 0.00, as shown by thePolyPhen-2 (Polymorphism Phenotyping v2) tool.G&gt;C transversion of intron variant in exon 21 of MYBPC3 results in a new exon splicing enhancer (ESE) site which may alter splicing. G&gt;A transition at g. 20167 is an intron variant of a previously reported status (rs373904644). This polymorphism results in the creation of an intronic splicingenhancer (ISE) site. The proband had left ventricular wall thickness of 24 mm, ejection fraction of 60 (%), and left ventricular internal diameter of 30 mm, suggesting early onset of HCM. Conclusion: The MYBPC3 mutations might play a key role in thedevelopment of HCM in the Indian population, indicating the soundness of establishing a systematic screening of all HCM patients.