Exonic Splicing Silencer Research Articles

Splicing factor hnRNPA1 regulates alternative splicing of LOXL2 to enhance the production of LOXL2Δ13

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family and has the ability to catalyze the cross-linking of extracellular matrix collagen and elastin. High expression of LOXL2 is related to tumor cell proliferation, invasion and metastasis. LOXL2 contains 14 exons. Previous studies have found that LOXL2 has abnormal alternative splicing and exon skipping in a variety of tissues and cells, resulting in a new alternatively-spliced isoform denoted LOXL2Δ13. LOXL2Δ13 lacks LOXL2WT exon 13, but its encoded protein has greater ability to induce tumor cell proliferation, invasion and metastasis. However, the molecular events that produce LOXL2Δ13 are still unclear. In this study, we found that overexpression of the splicing factor hnRNPA1 in cells can regulate the alternative splicing of LOXL2 and increase the expression of LOXL2Δ13. The exonic splicing silencer (ESS) exists at the 3′ splice site (3′ SS) and 5′ splice site (5′ SS) of LOXL2 exon 13. HnRNPA1 can bind to the ESS and inhibit the inclusion of exon 13. The RRM domain of hnRNPA1 and phosphorylation of hnRNPA1 at S91 and S95 are important for the regulation of LOXL2 alternative splicing. These results show that hnRNPA1 is a splicing factor that enhances the production of LOXL2Δ13.

Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3.

Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear. The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells. HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well. Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.

Copper-binding proteins and exonic splicing enhancers and silencers.

Eukaryotic DNA codes not only for proteins but contains a wealth of information required for accurate splicing of messenger RNA precursors and inclusion of constitutively or alternatively spliced exons in mature transcripts. This "auxiliary" splicing code has been characterized as exonic splicing enhancers and silencers (ESE and ESS). The exact interplay between protein and splicing codes is, however, poorly understood. Here, we show that exons encoding copper-coordinating amino acids in human cuproproteins lack ESEs and/or have an excess of ESSs, yet RNA sequencing and expressed sequence tags data show that they are more efficiently included in mature transcripts by the splicing machinery than average exons. Their largely constitutive inclusion in messenger RNA is facilitated by stronger splice sites, including polypyrimidine tracts, consistent with an important role of the surrounding intron architecture in ensuring high expression of metal-binding residues during evolution. ESE/ESS profiles of codons and entire exons that code for copper-coordinating residues were very similar to those encoding residues that coordinate zinc but markedly different from those that coordinate calcium. Together, these results reveal how the traditional and auxiliary splicing motifs responded to constraints of metal coordination in proteins.

Identified eleven exon variants in PKD1 and PKD2 genes that altered RNA splicing by minigene assay

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common monogenic multisystem disease caused primarily by mutations in the PKD1 gene or PKD2 gene. There is increasing evidence that some of these variants, which are described as missense, synonymous or nonsense mutations in the literature or databases, may be deleterious by affecting the pre-mRNA splicing process.ResultsThis study aimed to determine the effect of these PKD1 and PKD2 variants on exon splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 19 candidate single nucleotide alterations, 11 variants distributed in PKD1 (c.7866C > A, c.7960A > G, c.7979A > T, c.7987C > T, c.11248C > G, c.11251C > T, c.11257C > G, c.11257C > T, c.11346C > T, and c.11393C > G) and PKD2 (c.1480G > T) were identified to result in exon skipping.ConclusionsWe confirmed that 11 variants in the gene of PKD1 and PKD2 affect normal splicing by interfering the recognition of classical splicing sites or by disrupting exon splicing enhancers and generating exon splicing silencers. This is the most comprehensive study to date on pre-mRNA splicing of exonic variants in ADPKD-associated disease-causing genes in consideration of the increasing number of identified variants in PKD1 and PKD2 gene in recent years. These results emphasize the significance of assessing the effect of exon single nucleotide variants in ADPKD at the mRNA level.

A newly identified β-amyrin synthase gene hypothetically involved in oleanane-saponin biosynthesis from Talinum paniculatum (Jacq.) Gaertn.

Talinum paniculatum or Javanese ginseng in Indonesia is a plant widely used as a traditional medicine. The genus Talinum produces oleanane-type saponins, such as talinumoside I. The first aim of this study was to isolate the probable gene encoding β-amyrin synthase (bAS), a key enzyme involved in the cyclization of 2,3-oxidosqualene producing the backbone of the oleanane-type saponin β-amyrin and characterize the gene sequence and the predicted protein sequence using in silico approach. The second aim was to analyze the correlation between the TpbAS gene expression level and saponin production in various plant organs. Thus, TpbAS was isolated using degenerate primers and PCR 5'/3′-Rapid Amplification of cDNA Ends (RACE), then the gene sequence and the predicted protein were in silico analyzed using various programs. TpbAS expression level was analyzed using reverse transcriptase PCR (RT-PCR), and saponin content was measured using a spectrophotometer. The results showed that the full-length TpbAS gene consists of 2298 base pairs encoding for a 765-amino acid protein. From in silico study, the (GA)n sequence was identified in the 5′-untranslated regions and predicted to be a candidate of the gene expression modulator. In addition, functional RNA motifs and sites analysis predicted the presence of exon splicing enhancers and silencers within the coding sequence and miRNA target sites candidate. Amino acid sequence analysis showed DCTAE, QW, and WCYCR motifs that were conserved in all classes of oxidosqualene cyclase enzymes. Phylogenetic tree analysis showed that TpbAS is closely related to other plant oxidosqualene cyclase groups. Analysis of TpbAS expression and saponin content indicated that saponin is mainly synthesized and accumulated in the leaves. Taken together, these findings will assist in increasing the saponin content through a metabolic engineering approach.

Haplotypic variants of COVID-19 related genes are associated with blood pressure and metabolites levels.

The genetic association of coronavirus disease 2019 (COVID-19) with its complications has not been fully understood. This study aimed to identify variants and haplotypes of candidate genes implicated in COVID-19 related traits by combining the literature review and pathway analysis. To explore such genes, the protein-protein interactions and relevant pathways of COVID-19-associated genes were assessed. A number of variants on candidate genes were identified from Genome-wide association studies (GWASs) which were associated with COVID-19 related traits (p ˂ 10-6 ). Haplotypic blocks were assessed using haplotypic structures among the 1000 Genomes Project (r2 ≥ 0.8, D' ≥ 0.8). Further functional analyses were performed on the selected variants. The results demonstrated that a group of variants in ACE and AGT genes were significantly correlated with COVID-19 related traits. Three haplotypes were identified to be involved in the blood metabolites levels and the development of blood pressure. Functional analyses revealed that most GWAS index variants were expression quantitative trait lociand had transcription factor binding sites, exonic splicing enhancers or silencer activities. Furthermore, the proxy haplotype variants, rs4316, rs4353, rs4359, and three variants, namely rs2493133, rs2478543, and rs5051, were associated with blood metabolite and systolic blood pressure, respectively. These variants exerted more regulatory effects compared with other GWAS variants. The present study indicates that the genetic variants and candidate haplotypes of COVID-19 related genes are associated with blood pressure and blood metabolites. However, further observational studies are warranted to confirm these results.

A splicing silencer in SMN2 intron 6 is critical in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a fatal neuromuscular disease caused by homozygous deletions or mutations of the SMN1 gene. SMN2 is a paralogous gene of SMN1 and a modifying gene of SMA. A better understanding of how SMN2 exon 7 splicing is regulated helps discover new therapeutic targets for SMA therapy. Based on an antisense walk method to map exonic and intronic splicing silencers (ESSs and ISSs) in SMN2 exon 7 and the proximal regions of its flanking introns, we identified one ISS (ISS6-KH) at upstream of the branch point site in intron 6. By using mutagenesis-coupled RT-PCR with SMN1/2 minigenes, immunochromatography, overexpression and siRNA-knockdown, we found this ISS consists of a bipartite hnRNP A1 binding cis-element and a poly-U sequence located between the proximal hnRNP A1 binding site (UAGCUA) and the branch site. Both HuR and hnRNP C1 proteins promote exon 7 skipping through the poly-U stretch. Mutations or deletions of these motifs lead to efficient SMN2 exon 7 inclusion comparable to SMN1 gene. Furthermore, we identified an optimal antisense oligonucleotide that binds the intron six ISS and causes striking exon 7 inclusion in the SMN2 gene in patient fibroblasts and SMA mouse model. Our findings demonstrate that this novel ISS plays an important role in SMN2 exon 7 skipping and highlight a new therapeutic target for SMA therapy.

Position-dependent effects of hnRNP A1/A2 in SMN1/2 exon7 splicing

Heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNP A1/2) is a ubiquitously expressed RNA binding protein known to bind intronic or exonic splicing silencer. Binding of hnRNP A1/2 to survival of motor neuron gene (SMN1/2) exon 7 and flanking sequences strongly inhibits the inclusion of exon 7, which causes spinal muscular atrophy, a common genetic disorder. However, the role of hnRNP A1/2 on the side away from exon 7 is unclear. Here using antisense oligonucleotides, we fished an intronic splicing enhancer (ISE) near the 3′-splice site (SS) of intron 7 of SMN1/2. Mutagenesis identified the efficient motif of the ISE as “UAGUAGG”, coupled with RNA pull down and protein overexpression, we proved that hnRNP A1/2 binding to the ISE promotes the inclusion of SMN1/2 exon 7. Using MS2-tethering array and “UAGGGU” motif walking, we further uncovered that effects of hnRNP A1/2 on SMN1/2 exon 7 splicing are position-dependent: exon 7 inclusion is inhibited when hnRNP A1/2 binds proximal to the 5′SS of intron 7, promoted when its binds proximal to the 3′SS. These data provide new insights into the splicing regulatory mechanism of SMN1/2.

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.

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.

Encoded Conformational Dynamics of the HIV Splice Site A3 Regulatory Locus: Implications for Differential Binding of hnRNP Splicing Auxiliary Factors

Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base–base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3′ end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus.

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.

Genetic variation in phenylketonuria: analysis of the PAHvdb database

Introduction. Phenylketonuria (PKU) is the most frequent inborn metabolism error. The principal determinant factor for the metabolic phenotype in PKU is the residual enzymatic activity, which is determined by the variations in the phenylalanine hydroxylase (PAH) gene. To date, there are known over 1200 PAH gene variants contained in the PAH International Database of Variations in Phenylalanine Hydroxylase Gene (PAHvdb). Aim. The aim of this study is to elaborate an updated PAH variant report with their type, localization, frequency and severity. Material and method. The PAH variant analysis was made using PAHvdb. PAHvdb presently contains 1285 PAH variants and is connected to BIOPKU genotype-phenotype database which has anonymized information from nearly 18000 PKU patients, with data regarding the genotype and the correspondent phenotype. Results. From the 1285 studied variants, the most frequent variants are substitutions – 1051 (81.8%) and deletions – 150 (11.7 %). The majority (723 - 56.3%) is represented by missense mutations, followed in frequency by frameshift variants - 177 (13.8%), splice ESS (Exonic Splicing Silencer) variants - 127 (9.9%) and nonsense mutations - 86 (6.7%). The most affected region of the gene is exon 6 (169 variants), followed by exon 7 (151 variants), exon 3 (130 variants) and exon 11 (121 variants). The majority of variants are located in the catalytic (57.66%) domain. The most frequent 3 alleles are c.1222C>T with a frequency of 19,2%, c.1066-11G>A with a frequency of 6,8% and c.782G>A with a frequency of 5.5%. From the total 1285 variants, 488 (38.5%) cause a severe phenotype, 57 (4.9%) cause a moderate phenotype and 74 (6%) cause a mild phenotype; in the case of 666 variants (50,6%) which have a low allelic frequency the metabolic phenotype couldn’t be established. Discussions. The majority of the variants are substitutions, missense in the catalytic domain of the gene, on the 6th exon. Approximatively 50% of the alleles are found in single patients so they can’t be used for phenotypic prediction. The majority of variants are causing a severe phenotype. Conclusion. The existence of a database with a large number of PKU patients and PAH variants brings an important contribution to the understanding of the genotype-phenotype relation and to the capacity of better phenotypical prediction based on genotype.

Vulnerable exons, like ACADM exon 5, are highly dependent on maintaining a correct balance between splicing enhancers and silencers.

It is now widely accepted that aberrant splicing of constitutive exons is often caused by mutations affecting cis-acting splicing regulatory elements, but there is a misconception that all exons have an equal dependency on splicing regulatory elements and thus a similar susceptibility to aberrant splicing. We investigated exonic mutations in ACADM exon 5 to experimentally examine their effect on splicing and found that 7 out of 11 tested mutations affected exon inclusion, demonstrating that this constitutive exon is particularly vulnerable to exonic splicing mutations. Employing ACADM exon 5 and 6 as models, we demonstrate that the balance between splicing enhancers and silencers, flanking intron length, and flanking splice site strength are important factors that determine exon definition and splicing efficiency of the exon in question. Our study shows that two constitutive exons in ACADM have different inherent vulnerabilities to exonic splicing mutations. This suggests that in silico prediction of potential pathogenic effects on splicing from exonic mutations may be improved by also considering the inherent vulnerability of the exon. Moreover, we show that single nucleotide polymorphism that affect either of two different exonic splicing silencers, located far apart in exon 5, all protect against both immediately flanking and more distant exonic splicing mutations.

Identification and in silico characterization of structural and functional impacts of genetic variants in milk protein genes in the Zebu breeds Guzerat and Gyr.

Whole genome sequencing of bovine breeds has allowed identification of genetic variants in milk protein genes. However, functional repercussion of such variants at a molecular level has seldom been investigated. Here, the results of a multistep Bioinformatic analysis for functional characterization of recently identified genetic variants in Brazilian Gyr and Guzerat breeds is described, including predicted effects on the following: (i) evolutionary conserved nucleotide positions/regions; (ii) protein function, stability, and interactions; (iii) splicing, branching, and miRNA binding sites; (iv) promoters and transcription factor binding sites; and (v) collocation with QTL. Seventy-one genetic variants were identified in the caseins (CSN1S1, CSN2, CSN1S2, and CSN3), LALBA, LGB, and LTF genes. Eleven potentially regulatory variants and two missense mutations were identified. LALBA Ile60Val was predicted to affect protein stability and flexibility, by reducing the number the disulfide bonds established. LTF Thr546Asn is predicted to generate steric clashes, which could mildly affect iron coordination. In addition, LALBA Ile60Val and LTF Thr546Asn affect exonic splicing enhancers and silencers. Consequently, both mutations have the potential of affecting immune response at individual level, not only in the mammary gland. Although laborious, this multistep procedure for classifying variants allowed the identification of potentially functional variants for milk protein genes.

Combination targeted treatment may enhance antitumor activity in ERBB3 amplified high-grade serous endometrial cancer cells resistant to single agent targeted therapy

Objectives: Up-regulation of ERBB pathways represent targetable alterations in many high grade endometrial cancers (EC); ERBB3 amplifications in particular may contribute to ineffective pathway targeting due to persistent co-activation of other ERBB binding partners, leading to tumor growth and survival. The efficacy of downstream dual-inhibition of MEK+AKT or MEK+PI3K in an ERBB3 amplified EC primary tumor was studied using a microcancer 3D ex-vivo tumor cell viability assay. Methods: Tumor was prospectively collected from a patient with stage II, FIGO grade 3, serous EC and genomic characterization was performed using multiple sequencing methods: whole exome, RNA, and MatePair analysis. Somatic mutations, structural variance, with transcriptomic profiling was used to identify potential driver pathways for subsequent pharmacologic inhibition. Primary tumor cells were grown in a three-dimensional environment and the formed microcancers were subjected to drug treatment. Cell viability was determined by the CellTiter-Glow Luminescent Assay. Data transformation and dose-response curves were generated using GraphPad PRISM using four parameter logistic regression. CompuSyn software with the Chou-Talalay method was used to analyze drug interactions and synergy. The Fractional response-Combination index (Fa-CI) plot was generated with Microsoft Excel. Capivasertib, GDC-0084, and cobimetinib were used to inhibit AKT, PI3K/mTOR, and MEK, respectively. Inhibitory effect was defined as percent reduction in ATP at clinically-defined predicted plasma maximum concentration (Cmax) values. Results: Genomic sequencing revealed overexpression of a mutated ERBB3 (c.889G>T; p.Asp297Tyr) transcript within a 6N focal amplification. Additional pertinent alterations included a MYC mid-level gain, and TP53 double hit (loss and exonic splicing silencer (ESS)). Inhibition of cell viability was moderate by single agents: capivasertib, cobimetanib, or GDC-0084, as shown by inhibitory effect values of 33.9%, 35.9% and 49.9%, respectively. Two combinations with cobimetinib demonstrated increasing inhibitory effect values of 76.2% for cobimetanib/capivasertib at the Cmax of capivasertib and 79.7% for cobimetanib/GDC-0084 at the Cmax of cobimetanib. Synergy was evidenced by a combination index <1 at all points with Fa>0.8 (Figure 1). Conclusions: Combined inhibition of MEK and PI3K-AKT-mTOR pathways synergistically suppress viability in a patient-derived high-grade serous EC harboring a ERBB3 amplification. Further ex-vivo testing should be performed in endometrial tumors with other ERBB3 aberrations to allow generalization of results. Up-regulation of ERBB pathways represent targetable alterations in many high grade endometrial cancers (EC); ERBB3 amplifications in particular may contribute to ineffective pathway targeting due to persistent co-activation of other ERBB binding partners, leading to tumor growth and survival. The efficacy of downstream dual-inhibition of MEK+AKT or MEK+PI3K in an ERBB3 amplified EC primary tumor was studied using a microcancer 3D ex-vivo tumor cell viability assay. Tumor was prospectively collected from a patient with stage II, FIGO grade 3, serous EC and genomic characterization was performed using multiple sequencing methods: whole exome, RNA, and MatePair analysis. Somatic mutations, structural variance, with transcriptomic profiling was used to identify potential driver pathways for subsequent pharmacologic inhibition. Primary tumor cells were grown in a three-dimensional environment and the formed microcancers were subjected to drug treatment. Cell viability was determined by the CellTiter-Glow Luminescent Assay. Data transformation and dose-response curves were generated using GraphPad PRISM using four parameter logistic regression. CompuSyn software with the Chou-Talalay method was used to analyze drug interactions and synergy. The Fractional response-Combination index (Fa-CI) plot was generated with Microsoft Excel. Capivasertib, GDC-0084, and cobimetinib were used to inhibit AKT, PI3K/mTOR, and MEK, respectively. Inhibitory effect was defined as percent reduction in ATP at clinically-defined predicted plasma maximum concentration (Cmax) values. Genomic sequencing revealed overexpression of a mutated ERBB3 (c.889G>T; p.Asp297Tyr) transcript within a 6N focal amplification. Additional pertinent alterations included a MYC mid-level gain, and TP53 double hit (loss and exonic splicing silencer (ESS)). Inhibition of cell viability was moderate by single agents: capivasertib, cobimetanib, or GDC-0084, as shown by inhibitory effect values of 33.9%, 35.9% and 49.9%, respectively. Two combinations with cobimetinib demonstrated increasing inhibitory effect values of 76.2% for cobimetanib/capivasertib at the Cmax of capivasertib and 79.7% for cobimetanib/GDC-0084 at the Cmax of cobimetanib. Synergy was evidenced by a combination index <1 at all points with Fa>0.8 (Figure 1). Combined inhibition of MEK and PI3K-AKT-mTOR pathways synergistically suppress viability in a patient-derived high-grade serous EC harboring a ERBB3 amplification. Further ex-vivo testing should be performed in endometrial tumors with other ERBB3 aberrations to allow generalization of results.

Identification of a Novel Splice Site Mutation in RUNX2 Gene in a Family with Rare Autosomal Dominant Cleidocranial Dysplasia.

Background:Pathogenic variants of RUNX2, a gene that encodes an osteoblast-specific transcription factor, have been shown as the cause of CCD, which is a rare hereditary skeletal and dental disorder with dominant mode of inheritance and a broad range of clinical variability. Due to the relative lack of clinical complications resulting in CCD, the medical diagnosis of this disorder is challenging, which leaves it underdiagnosed. Methods:In this study, nine healthy and affected members of an Iranian family were investigated. PCR and sequencing of all exons and exon-intron boundaries of RUNX2 (NM_001024630) gene was performed on proband. Co-segregation analysis was conducted in the other family members for the identified variant. Additionally, a cohort of 100 Iranian ethnicity-matched healthy controls was screened by ARMS-PCR method. Results:The novel splice site variant (c.860-2A>G), which was identified in the intron 6 of RUNX2 gene, co-segregated with the disease in the family, and it was absent in healthy controls. Pathogenicity of this variant was determined by several software, including HSF, which predicts the formation or disruption of splice donor sites, splice acceptor sites, exonic splicing silencer sites, and exonic splicing enhancer sites. In silico analysis predicted this novel variant to be disease causing. Conclusion:The identified variant is predicted to have an effect on splicing, which leads to exon skipping and producing a truncated protein via introducing a premature stop codon.

Identification of seven exonic variants in the SLC4A1, ATP6V1B1, and ATP6V0A4 genes that alter RNA splicing by minigene assay.

Primary distal renal tubular acidosis (dRTA) is a rare tubular disease associated with variants in SLC4A1, ATP6V0A4, ATP6V1B1, FOXⅠ1, or WDR72 genes. Currently, there is growing evidence that all types of exonic variants can alter splicing regulatory elements, affecting the precursor messenger RNA (pre-mRNA) splicing process. This study was to determine the consequences of variants associated with dRTA on pre-mRNA splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 15 candidate variants, 7 variants distributed in SLC4A1 (c.1765C>T, p.Arg589Cys), ATP6V1B1 (c.368G>T, p.Gly123Val; c.370C>T, p.Arg124Trp; c.484G>T, p.Glu162* and c.1102G>A, p.Glu368Lys) and ATP6V0A4 genes (c.322C>T, p.Gln108* and c.1572G>A, p.Pro524Pro) were identified to result in complete or incomplete exon skipping by either disruption of exonic splicing enhancers (ESEs) and generation of exonic splicing silencers, or interference with the recognition of the classic splicing site, or both. To our knowledge, this is the first study on pre-mRNA splicing of exonic variants in the dRTA-related genes. These results highlight the importance of assessing the effects of exonic variants at the mRNA level and suggest that minigene analysis is an effective tool for evaluating the effects of splicing on variants in vitro.

Mutation of P-element somatic inhibitor induces male sterility in the diamondback moth, Plutella xylostella.

Genetic manipulation of sex determination pathways in insects provides the basis for a broad range of strategies to benefit agricultural security and human health. The P-element somatic inhibitor (PSI) protein, an exon splicing silencer that promotes male-specific splicing of dsx, plays a critical role in male sexual differentiation and development. The functions of PSI have been characterized in the lepidopteran model species Bombyx mori. However, the molecular mechanism and functions of PSI in Plutella xylostella, a worldwide agricultural pest and taxonomically basal species, are still unknown. Here we identified PxPSI transcripts and analyzed their spatiotemporal expression pattern in P. xylostella. Multiple sequence alignment revealed that PxPSI contains four KH domains and is highly conserved in lepidopterans. We used the CRISPR-Cas9 system to generate mutations of the PxPSI genomic locus. Disruptions of PxPSI caused male-specific defects in internal and external genitals. In addition, we detected female-specific Pxdsx transcripts in PxPSI male mutants. Mutations also caused changes in expression of several sex-biased genes and induced male sterility. Our study demonstrates that PxPSI plays a key role in male sex determination in P. xylostella and suggests a potential molecular target for genetic-based pest management in lepidopteran pests. © 2021 Society of Chemical Industry.

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.