Exonic Splicing Enhancer Research Articles

Exploring Splicing Factor Aberrations As a Therapeutic Target in Mature B-Cell Malignancies

Objectives Cancers originating from mature B cells at various stages of terminal differentiation represent a diverse set of cancers. There are numerous pathological subtypes, each comprising genetically and clinically heterogeneous malignancies with distinct biology and clinical outcomes. We have previously identified the recurrence of mutations in splicing factor HNRNPH1 in mantle cell lymphoma (MCL), which were associated with inferior outcomes. Based on growing evidence for the role of splicing factors in mature B cell neoplasms, we sought to extend our characterization of splicing factor aberrations across additional B cell malignancies. We hypothesize that regulation of alternative splicing is essential for B cell differentiation and maturation, and aberrations of splicing factors in B cell malignancies are related to these normal patterns of alternative splicing. Ultimately, we are pursuing alternative splicing as a potential novel therapeutic vulnerability common among mature B cell neoplasms. Methods We leveraged whole genome, exome, and RNA sequencing data from 2999 tumors assembled from controlled-access repositories and ongoing projects at BC Cancer as part of our Genomic Analysis of Mature B-cell Lymphomas (GAMBL) project. RNA-seq data was aligned with STAR and splicing events were quantified with RMATS. Novel splicing events were quantified following de novo transcriptome assembly with StringTie. Simple somatic mutations were detected using a workflow comprised of four algorithms to identify high-confidence variants. We used additional RNA-seq data from distinct B-cell maturation states (naïve, centroblast, centrocyte, memory, bone marrow plasma, and tonsillar plasma cells) to identify annotated and novel splicing events which are differentially regulated throughout B cell maturation. Annotated and novel events were subsequently used to query alternative splicing in a subset of tumor samples using a combination of Illumina-based and Oxford Nanopore sequencing. Results More than 30% of patient samples harboured somatic mutations in at least one of 67 queried splicing factors. HNRNPU, PCBP1, SF3B1, DAZAP1, and HNRNPH1 were most commonly mutated, 13% of cases (Figure 1A). Although only reported in MCL thus far, we noted an abundance of HNRNPH1 mutations in DLBCL showing the same pattern of non-coding mutations, most commonly in the activated B cell subgroup. In non-malignant samples, we identified 129 known and 267 novel splicing events which display differential regulation during B cell maturation and warrant further investigation within subtypes of B cell malignancies (Figure 1B). Based on this observation, we hypothesize that the RNA isoform repertoire may vary across B cell differentiation states and its perturbation may contribute to oncogenesis in a context-dependent manner. Furthermore, the importance of alternative splicing during B cell maturation implies that perturbation of splicing in B cell malignancies could play a role in pathogenesis. We previously demonstrated that non-coding mutations in HNRNPH1 contribute to overexpression of the hnRNP H protein and are associated with poor patient outcomes in MCL. Based on the predominance of these mutations in DLBCL, we sought to evaluate hnRNP H overexpression as a potential therapeutic target. We identified a putative exonic splicing enhancer (ESE) with minimal secondary structure in the essential exon of HNRNPH1 using ESEfinder and designed locked nucleic acid antisense oligos (ASOs) complementary to the ESE. Using ddPCR, we showed that the ESE ASO can effectively decrease the proportion of the productive HNRNPH1 splice variant at nanomolar concentrations in HEK cells; however, a splice site ASO does not display the same efficacy. Conclusions We have identified a growing number of splicing factors in mature B cell malignancies as recurrently mutated, indicating that deregulation of the RNA isoform repertoire in B cells can contribute to oncogenesis. Characterizing the landscape of alternative splicing in normal and malignant B cells will allow us to identify opportunities to exploit this reliance in the development of novel therapeutic strategies. Our data highlight antisense oligonucleotides as a promising therapeutic avenue for targeting splicing aberrations caused by common mutations in B cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Creatine Deficiency Syndromes: Comparison of Screening Methods and Characterization of Four Novel Intronic Variants

BackgroundCerebral creatine deficiency syndromes (CCDS) are disorders affecting creatine synthesis or transport. Several methods have been developed to measure creatine and guanidinoacetate (GAA) in different body fluids including methods based on gas chromatography-mass spectrometry (GC–MS) and High-pressure liquid chromatography mass spectrometry (HPLC-MS). The diagnosis of CCDS is then confirmed by sequencing of creatine biosynthesis genes guanidinoacetate methyltransferase (GAMT) and Arginine: glycine amidinotransferase (GATM) and creatine transporter gene solute carrier family 6 member 8 (SLC6A8) or by functional enzymatic assay. The aim of the current study was to find the most reliable and accurate screening method for CCDS by comparing methods using Nuclear Magnetic Resonance spectroscopy (NMR), GC–MS and HPLC-MS. Additionally, this study was performed to estimate the prevalence of CCDS in a cohort of Egyptian patients and potentially to discover novel variants. Subjects and methodsThe study was conducted on 150 subjects with clinical signs and symptoms consistent with CCDS. Metabolic profiling of urine samples was performed using three techniques: 1) GC–MS 2) Ultra high-pressure (or performance) liquid chromatography – Tandem Mass Spectrometry (UHPLC- MS/MS) and 3) NMR. ResultsThe linearity of peak areas for creatine and GAA by UHPLC-MS/MS and NMR covered and exceeded the ranges normally found in urine. The limit of quantification and the inter-day precision results for creatine and GAA were more robust by UHPLC-MS/MS than NMR. Ten cases were identified as being positive for CCDS by our analytical approaches and underwent next generation sequencing (NGS) for GAMT, GATM and SLC6A8 genes. NGS was performed and confirmed one patient with one likely Pathogenic variant in GAMT gene: (NC_000019.10:g.1401317C > G, NP_000147.1:p.Ala54Pro). Additionally, we describe four novel intronic variants in the GATM gene: c.1043-357del and c.1043-357_1043-356insT, and were predicted to activate cryptic acceptor site with potential alteration of splicing, c.979-227G > A was found to significantly alter the Exon Splice Enhancer (ESE) xon Splice Silencer (ESS) motifs ratio and c.1042 + 262del which was found to have no implications on splicing. ConclusionsBoth UHPLC-MS/MS and NMR spectroscopy are comparable to GC–MS in screening for CCDS. Nonetheless, the UHPLC-MS/MS method had better performance than NMR spectroscopy. Additionally, Sequencing of the full length of GATM, GAMT, and SLC6A8 genes is needed to identify intronic variants that could cause CCDS via affecting splice sites.

Twelve exonic variants in the SLC12A1 and CLCNKB genes alter RNA splicing in a minigene assay.

Background: Bartter syndrome (BS) is a rare renal tubular disease caused by gene variants in SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND or MAGED2 genes. There is growing evidence that many exonic mutations can affect the pre-mRNA normal splicing and induce exon skipping by altering various splicing regulatory signals. Therefore, the aim of this study was to gain new insights into the consequences of exonic mutations associated with BS on pre-mRNA splicing. Methods: We analyzed all the missense, nonsense and synonymous variants described in six pathogenic genes by bioinformatics programs and identified candidate mutations that may promote exon skipping through a minigene system. Results: Results of the study showed that 12 of 14 candidate variants distributed in SLC12A1 (c.728G>A, C.735C>G, c.904C>T, c.905G>A, c.1304C>T, c.1493C>T, c.2221A>T) and CLCNKB (c.226C>T, c.228A>C, c.229G>A, c.229G>C, c.1979C>A) were identified to induce splicing alterations. These variants may not only disrupt exonic splicing enhancers (ESEs) but also generate new exonic splicing silencers (ESSs), or disturb the classic splicing sites. Conclusion: To our knowledge, this is a comprehensive study regarding alterations in pre-mRNA of exonic variants in BS pathogenic genes. Our results reinforce the necessity of assessing the consequences of exonic variants at the mRNA level.

Alternatively spliced CSF3R isoforms in SRSF2 P95H mutated myeloid neoplasms.

Alternatively spliced colony stimulating factor 3 receptor (CSF3R) isoforms Class III and Class IV are observed in myelodysplastic syndromes (MDS), but their roles in disease remain unclear. We report that the MDS-associated splicing factor SRSF2 affects the expression of Class III and Class IV isoforms and perturbs granulopoiesis. Add-back of the Class IV isoform in Csf3r-null mouse progenitor cells increased granulocyte progenitors with impaired neutrophil differentiation, while add-back of the Class III produced dysmorphic neutrophils in fewer numbers. These CSF3R isoforms were elevated in patients with myeloid neoplasms harboring SRSF2 mutations. Using in vitro splicing assays, we confirmed increased Class III and Class IV transcripts when SRSF2 P95 mutations were co-expressed with the CSF3R minigene in K562 cells. Since SRSF2 regulates splicing partly by recognizing exonic splicing enhancer (ESE) sequences on pre-mRNA, deletion of either ESE motifs within CSF3R exon 17 decreased Class IV transcript levels without affecting Class III. CD34+ cells expressing SRSF2 P95H showed impaired neutrophil differentiation in response to G-CSF and was accompanied by increased levels of Class IV. Our findings suggest that SRSF2 P95H promotes Class IV splicing by binding to key ESE sequences in CSF3R exon 17, and that SRSF2, when mutated, contributes to dysgranulopoiesis.

A missense mutation in TTC8/BBS8 affecting mRNA splicing in patients with non-syndromic retinitis pigmentosa.

Splicing disruption is one type of mutation mechanism for disease-predisposing alleles. To date, less than 30 mutations in TTC8/BBS8 have been reported; however, mutations affecting the splice site are rare. Generally missense mutations are assumed to alter protein function; however, reports have shown that mutations in protein coding exons can disrupt splicing by altering exonic splicing silencer or enhancer motifs. Hence, a missense mutation c.1347G > C (p.Q449H) involving final base of the exon 13 in the TTC8, previously identified by us to be linked with non-syndromic autosomal recessive retinitis pigmentosa (arRP), in an Indian family, that might deleteriously affect splicing has been functionally characterized. RNA was isolated, cDNA prepared and amplified using region-specific primers. PCR products were purified and sequenced bi-directionally by Sanger sequencing. Effect of mutation (c.1347G > C) on mRNA splicing has been predicted using bioinformatics tools. We reported that missense mutation (c.1347G > C) at the last base of exon 13 of TTC8 disrupted the canonical donor splice-site resulting in aberrant RNA splicing. A cryptic donor splice-site got activated 77 bases downstream of the authentic splice donor site in intron 13, resulting in the retention of 77 bases of intron 13, and a frameshift leading to pre-mature termination codon in exon 14 at codon 486. Further, duplication of exon 15 and fusion of its duplicated copy occurred with exon 13. The binding site for SC35 protein, normally involved in splicing, also got disrupted (as predicted by SpliceAid2 software), hence, leading to alternative splicing. Our findings strongly suggest that a missense mutation c.1347G > C in TTC8 disrupted the splice donor site causing retention of 77 bases of intron 13, resulting in a frameshift and subsequently introduced a pre-mature termination codon into exon 14, hence creating an altered mRNA transcript. These findings emphasize the significance of examining missense mutations especially in TTC8, to determine their pathogenic role through alternative splicing. Present findings also reiterate the notion that mutations in the TTC8/BBS8 cause phenotypic heterogeneity and does not always follow Mendelian genetics in this ciliopathy.

Recognition of functional genetic polymorphism using ESE motif definition: a conservative evolutionary approach to CYP2D6/CYP2C19 gene variants.

Although predicting the effects of variants near intron-exon boundaries is relatively straightforward, predicting the functional Exon Splicing Enhancers (ESEs) and the possible effects of variants within ESEs remains a challenge. Considering the essential role of CYP2D6/CYP2C19 genes in drug metabolism, we attempted to identify variants that are most likely to disrupt splicing through their effect on these ESEs. ESEs were predicted in these two genes using ESEfinder 3.0, incorporating a series of filters (increased threshold and evolutionary conservation). Finally, reported mutations were evaluated for their potential to disrupt splicing by affecting these ESEs. Initially, 169 and 243 ESEs were predicted for CYP2C19/CYP2D6, respectively. However, applying the filters, the number of predicted ESEs was reduced to 26 and 19 in CYP2C19/CYP2D6, respectively. Comparing prioritized predicted ESEs with known sequence variants in CYP2C19/CYP2D6 genes highlights 18 variations within conserved ESEs for each gene. We found good agreement in cases where such predictions could be compared to experimental evidence. In total, we prioritized a subset of mutational changes in CYP2C19/CYP2D6 genes that may affect the function of these genes and lead to altered drug responses. Clinical studies and functional analysis for investigating detailed functional consequences of the mentioned mutations and their phenotypic outcomes is mostly recommended.

SRPK1 regulates RNA binding in a pre-spliceosomal complex using a catalytic bypass mechanism.

Serine-arginine protein kinase 1 (SRPK1) phosphorylates serine-arginine (SR) proteins in the cytoplasm, directing them to the nucleus for splicing function. SRPK1 has also been detected in the nucleus but its function here is still not fully understood. We now demonstrate that nuclear SRPK1 can regulate U1-70K, a protein component of the uridine-rich 1 small nuclear ribonucleoprotein (U1 snRNP) that binds SR proteins and facilitates 5' splice-site selection in precursor mRNA. We found that SRPK1 uses a large, disordered domain to bind U1-70K, regulating the interaction of an exonic splicing enhancer (ESE) to the associated SR protein. Surprisingly, the catalytic activity of SRPK1 is not required for this phenomenon. Instead, SRPK1 associates directly with the N-terminus of U1-70K and alters the regulatory function of the distal C-terminus, modifying interactions between the U1-70K:SR protein complex and the ESE. Disruption of SRPK1 binding to this complex affects the alternative splicing of genes modulated by the C-terminus of U1-70K. Such findings suggest that, in addition to operating as a traditional serine-modifying catalyst, SRPK1 can also bypass this intrinsic activity to regulate RNA contacts in an early pre-spliceosomal complex.

Noncanonical Splice Site and Deep Intronic FRMD7 Variants Activate Cryptic Exons in X-linked Infantile Nystagmus.

PurposeWe aim to report noncoding pathogenic variants in patients with FRMD7-related infantile nystagmus (FIN).MethodsGenome sequencing (n = 2 families) and reanalysis of targeted panel next generation sequencing (n = 2 families) was performed in genetically unsolved cases of suspected FIN. Previous sequence analysis showed no pathogenic coding variants in genes associated with infantile nystagmus. SpliceAI, SpliceRover, and Alamut consensus programs were used to annotate noncoding variants. Minigene splicing assay was performed to confirm aberrant splicing. In silico analysis of exonic splicing enhancer and silencer was also performed.Results FRMD7 intronic variants were identified based on genome sequencing and targeted next-generation sequencing analysis. These included c.285-12A>G (pedigree 1), c.284+63T>A (pedigrees 2 and 3), and c. 383-1368A>G (pedigree 4). All variants were absent in gnomAD, and the both c.285-12A>G and c.284+63T>A variants were predicted to enhance new splicing acceptor gains with SpliceAI, SpliceRover, and Alamut consensus approaches. However, the c.383-1368 A>G variant only had a significant impact score on the SpliceRover program. The c.383-1368A>G variant was predicted to promote pseudoexon inclusion by binding of exonic splicing enhancer. Aberrant exonizations were validated through minigene constructs, and all variants were segregated in the families.ConclusionsDeep learning–based annotation of noncoding variants facilitates the discovery of hidden genetic variations in patients with FIN. This study provides evidence of effectiveness of combined deep learning–based splicing tools to identify hidden pathogenic variants in previously unsolved patients with infantile nystagmus.Translational RelevanceThese results demonstrate robust analysis using two deep learning splicing predictions and in vitro functional study can lead to finding hidden genetic variations in unsolved patients.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia

AbstractDetailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3′ end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3′ splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Effects of Inhibitors of SLC9A-Type Sodium-Proton Exchangers on Survival Motor Neuron 2 (SMN2) mRNA Splicing and Expression.

Spinal muscular atrophy (SMA) is an autosomal recessive, pediatric-onset disorder caused by the loss of spinal motor neurons, thereby leading to muscle atrophy. SMA is caused by the loss of or mutations in the survival motor neuron 1 (SMN1) gene. SMN1 is duplicated in humans to give rise to the paralogous survival motor neuron 2 (SMN2) gene. This paralog is nearly identical except for a cytosine to thymine transition within an exonic splicing enhancer element within exon 7. As a result, the majority of SMN2 transcripts lack exon 7 (SMNΔ7), which produces a truncated and unstable SMN protein. Since SMN2 copy number is inversely related to disease severity, it is a well established target for SMA therapeutics development. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs), has previously been shown to increase exon 7 inclusion and SMN protein levels in SMA cells. In this study, NHE inhibitors were evaluated for their ability to modulate SMN2 expression. EIPA as well as 5-(N,N-hexamethylene)amiloride (HMA) increase exon 7 inclusion in SMN2 splicing reporter lines as well as in SMA fibroblasts. The EIPA-induced exon 7 inclusion occurs via a unique mechanism that does not involve previously identified splicing factors. Transcriptome analysis identified novel targets, including TIA1 and FABP3, for further characterization. EIPA and HMA are more selective at inhibiting the NHE5 isoform, which is expressed in fibroblasts as well as in neuronal cells. These results show that NHE5 inhibition increases SMN2 expression and may be a novel target for therapeutics development. SIGNIFICANCE STATEMENT: This study demonstrates a molecular mechanism by which inhibitors of the sodium-protein exchanger increase the alternative splicing of SMN2 in spinal muscular atrophy cells. NHE5 selective inhibitors increase the inclusion of full-length SMN2 mRNAs by targeting TIA1 and FABP3 expression, which is distinct from other small molecule regulators of SMN2 alternative splicing. This study provides a novel means to increase full-length SMN2 expression and a novel target for therapeutics development.

Innovative in Silico Approaches for Characterization of Genes and Proteins.

Bioinformatics is an amalgamation of biology, mathematics and computer science. It is a science which gathers the information from biology in terms of molecules and applies the informatic techniques to the gathered information for understanding and organizing the data in a useful manner. With the help of bioinformatics, the experimental data generated is stored in several databases available online like nucleotide database, protein databases, GENBANK and others. The data stored in these databases is used as reference for experimental evaluation and validation. Till now several online tools have been developed to analyze the genomic, transcriptomic, proteomics, epigenomics and metabolomics data. Some of them include Human Splicing Finder (HSF), Exonic Splicing Enhancer Mutation taster, and others. A number of SNPs are observed in the non-coding, intronic regions and play a role in the regulation of genes, which may or may not directly impose an effect on the protein expression. Many mutations are thought to influence the splicing mechanism by affecting the existing splice sites or creating a new sites. To predict the effect of mutation (SNP) on splicing mechanism/signal, HSF was developed. Thus, the tool is helpful in predicting the effect of mutations on splicing signals and can provide data even for better understanding of the intronic mutations that can be further validated experimentally. Additionally, rapid advancement in proteomics have steered researchers to organize the study of protein structure, function, relationships, and dynamics in space and time. Thus the effective integration of all of these technological interventions will eventually lead to steering up of next-generation systems biology, which will provide valuable biological insights in the field of research, diagnostic, therapeutic and development of personalized medicine.

Silent but Not Harmless: A Synonymous SLC5A5 Gene Variant Leading to Dyshormonogenic Congenital Hypothyroidism.

BackgroundCongenital iodide transport defect (ITD) is an uncommon cause of dyshormonogenic congenital hypothyroidism characterized by the absence of active iodide accumulation in the thyroid gland. ITD is an autosomal recessive disorder caused by loss-of-function variants in the sodium/iodide symporter (NIS)-coding SLC5A5 gene.ObjectiveWe aimed to identify, and if so to functionally characterize, novel ITD-causing SLC5A5 gene variants in a cohort of five unrelated pediatric patients diagnosed with dyshormonogenic congenital hypothyroidism with minimal to absent 99mTc-pertechnetate accumulation in the thyroid gland.MethodsThe coding region of the SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of a novel synonymous variant were performed.ResultsSanger sequencing revealed a novel homozygous synonymous SLC5A5 gene variant (c.1326A>C in exon 11). In silico analysis revealed that the c.1326A>C variant is potentially deleterious for NIS pre-mRNA splicing. The c.1326A>C variant was predicted to lie within a putative exonic splicing enhancer reducing the binding of splicing regulatory trans-acting protein SRSF5. Splicing minigene reporter assay revealed that c.1326A>C causes exon 11 or exon 11 and 12 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variants p.G415_P443del and p.G415Lfs*32, respectively. Significantly, the frameshift variant p.G415Lfs*32 is predicted to be subjected to degradation by nonsense-mediated decay.ConclusionsWe identified the first exonic synonymous SLC5A5 gene variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape of the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.

RNA Therapeutics for Brain Tumors: Targeting Pre‐mRNA Splicing Motifs to Generate Therapeutic Gene Isoforms

Individuals diagnosed with glioblastoma multiforme (GBM) have a short life expectancy of 12‐15 months. This project is to develop therapies for effective and continuous drug delivery to the brain, targeting cancer‐driving genes. Tumor cell proliferation in GBM is often stimulated by epidermal growth factor receptor (EGFR) and is important for tumor cell survival. In our lab, we are developing RNA therapies to alter the splicing mechanism of EGFR to block its activation, thus stop tumor cell growth. Our approach uses an adeno‐associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre‐mRNA transcript. In this project, we cloned therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. We have found that our therapies have led to a shift in Intron 10A retention increasing alternative intronic polyadenylation generating a short soluble therapeutic decoy. Our antisense therapies targeted the EGFR pre‐mRNA transcript, specifically, regions of exon 10 and intron 10. Therapies were either designed to enhance recognition of the alternative intronic polyadenylation signal or block recognition of canonical exon 10/exon 11 splicing. We targeted the Exonic Splicing Enhancer (ESE) wild‐type 5’ Splice Site (wt‐5’SS) of exon 10, and the regions surrounding the alternative intronic polyadenylation signal. Each target showed a significant increase in intron retention. Targeting the ESE showed a 5‐fold increase in intron retention (p < 0.04), the wt‐5’SS was 15‐fold (p < 0.001), and the region surrounding the alternative intronic polyadenylation signal showed the strongest effect, with greater than 60‐fold increase in intron retention (p < 0.002). Currently, we are examining additional antisense therapies, as well as generation of the therapeutic soluble decoy protein using ELISA.

Evaluation of Suspected Autosomal Alport Syndrome Synonymous Variants.

Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.

Exon skipping induced by CRISPR-directed gene editing regulates the response to chemotherapy in non-small cell lung carcinoma cells

We have been developing CRISPR-directed gene editing as an augmentative therapy for the treatment of non-small cell lung carcinoma (NSCLC) by genetic disruption of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). NRF2 promotes tumor cell survival in response to therapeutic intervention and thus its disablement should restore or enhance effective drug action. Here, we report how NRF2 disruption leads to collateral damage in the form of CRISPR-mediated exon skipping. Heterogeneous populations of transcripts and truncated proteins produce a variable response to chemotherapy, dependent on which functional domain is missing. We identify and characterize predicted and unpredicted transcript populations and discover that several types of transcripts arise through exon skipping; wherein one or two NRF2 exons are missing. In one specific case, the presence or absence of a single nucleotide determines whether an exon is skipped or not by reorganizing Exonic Splicing Enhancers (ESEs). We isolate and characterize the diversity of clones induced by CRISPR activity in a NSCLC tumor cell population, a critical and often overlooked genetic byproduct of this exciting technology. Finally, gRNAs must be designed with care to avoid altering gene expression patterns that can account for variable responses to solid tumor therapy.

Silent Mutations Reveal a Novel Treatment for RASQ61 Cancers.

Silent mutations are needed for functional KRASQ61K and revealed exonic splicing enhancer (ESE) motif enhancement around RASQ61.

Transgene-design: a web application for the design of mammalian transgenes.

SummaryTransgene-design is a web application to help design transgenes for use in mammalian studies. It is predicated on the recent discovery that human intronless transgenes and native retrogenes can be expressed very effectively if the GC content at exonic synonymous sites is high. In addition, as exonic splice enhancers resident in intron containing genes may have different utility in intronless genes, these can be reduced or increased in density. Input can be a native gene or a commercially ‘optimised’ gene. The option to leave in the first intron and to protect or avoid other motifs is also permitted. Availability and implementationTransgene-design is based on a ruby for rails platform. The application is available at https://transgene-design.bath.ac.uk. The code is available under GNU General Public License from GitHub (https://github.com/smuehlh/transgenes).Supplementary information Supplementary data are available at Bioinformatics online.

Silent mutations reveal therapeutic vulnerability in RAS Q61 cancers.

RAS family members are the most frequently mutated oncogenes in human cancers. Although KRAS(G12C)-specific inhibitors show clinical activity in patients with cancer1-3, there are no direct inhibitors of NRAS, HRAS or non-G12C KRAS variants. Here we uncover the requirement of the silent KRASG60G mutation for cells to produce a functional KRAS(Q61K). In the absence of this G60G mutation in KRASQ61K, a cryptic splice donor site is formed, promoting alternative splicing and premature protein termination. A G60G silent mutation eliminates the splice donor site, yielding a functional KRAS(Q61K) variant. We detected a concordance of KRASQ61K and a G60G/A59A silent mutation in three independent pan-cancer cohorts. The region around RAS Q61 is enriched in exonic splicing enhancer (ESE) motifs and we designed mutant-specific oligonucleotides to interfere with ESE-mediated splicing, rendering the RAS(Q61) protein non-functional in a mutant-selective manner. The induction of aberrant splicing by antisense oligonucleotides demonstrated therapeutic effects in vitro and in vivo. By studying the splicing necessary for a functional KRAS(Q61K), we uncover a mutant-selective treatment strategy for RASQ61 cancer and expose a mutant-specific vulnerability, which could potentially be exploited for therapy in other genetic contexts.

Sarcomeric gene mutations in phenotypic positive hypertrophic cardiomyopathic patients in Indian population.

HCM is a monogenic cardiac disorder with a high risk of sudden cardiac death, heterogeneous phenotypic expression and genetic profile. HCM is expressed as autosomal dominant in fashion with the prevalence of 1:500 in the general population. The main objective of the current study was to unravel the mutation status in sarcomeric genes in urbanizing Pune population. HCM patients were recruited from Bharti hospital and Poona hospital and research centre, Pune after being screened by 2-D echocardiography. DNA was extracted from whole blood samples and PCR amplification was performed for selected exons from pre-selected genes, amplimers of >300 b.p were restriction digested and the SSCP technique was optimized for maximum result output. HCM patients shows the maximum prevalence of mitral regurgitation (23.3%) while the minimum prevalence was left auricular diameter (10%). Maximum variation spectrum was present in MYBPC3 genes as most of them were "benign" type as per Polyphen-2 tool status. Mutations in the MYH7 gene produce a prominent impact on splicing by the creation of a new SRP40 binding site (Exon Splicing Enhancer) as predicted by Human Splicing Finder 3.1. I736T mutation in the MYH7 gene results in replacement of β-strand by α-helix upstream from mutation site which may have a profound impact on protein tertiary structure as predicted by Polyphen-2 tool (probably damaging-1.00). Also, two 'novel' mutations and one 'novel' variation were reported in the present study. Thus, the MYBPC3 gene shows maximum mutation load among other sarcomeric genes. Double gene mutations do not represent much severe pathophysiology as compared to single gene mutated and genotypic negative HCM patients.

Genetic Control of Splicing at SIRPG Modulates Risk of Type 1 Diabetes.

Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes, where it acts by engaging its ligand, CD47. SIRPG, which encodes SIRPγ, contains a nonsynonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. SIRPG produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of SIRPG transcript isoforms. All of these transcript isoforms produced protein upon transient expression in vitro. However, CRISPR/Cas9 targeting of one of the alternatively spliced exons in SIRPG eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild-type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling, and had significantly increased levels of cell-surface CD47. In primary CD4+ and CD8+ T cells, cell-surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that SIRPG is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.