Single Splice Research Articles

SPLICER: a highly efficient base editing toolbox that enables in vivo therapeutic exon skipping

Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which have broad applications in medicine and biotechnology. Existing techniques including antisense oligonucleotides, targetable nucleases, and base editors, while effective for specific applications, remain hindered by transient effects, genotoxicity, and inconsistent exon skipping. To overcome these limitations, here we develop SPLICER, a toolbox of next-generation base editors containing near-PAMless Cas9 nickase variants fused to adenosine or cytosine deaminases for the simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing improves exon skipping, reduces aberrant splicing, and enables skipping of exons refractory to single splice site editing. To demonstrate the therapeutic potential of SPLICER, we target APP exon 17, which encodes amino acids that are cleaved to form Aβ plaques in Alzheimer’s disease. SPLICER reduces the formation of Aβ42 peptides in vitro and enables efficient exon skipping in a mouse model of Alzheimer’s disease. Overall, SPLICER is a widely applicable and efficient exon skipping toolbox.

Rare occurrence of cryptic 5' splice sites by downstream 3' splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana.

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta.

Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA.

The maturation, development, and function of regulatory T cells (Tregs) are under the control of the crucial transcription factor Forkhead Box Protein 3 (FoxP3). Through alternative splicing, the human FoxP3 gene produces four different splice variants: a full-length variant (FL) and truncated variants with deletions of each of exons 2 (∆2 variant) or 7 (∆7 variant) or a deletion of both exons (∆2∆7 variant). Their involvement in the biology of Tregs as well as their association with autoimmune diseases remains to be clarified. The aim of this work was to induce a single FoxP3 splice variant in human Tregs by splice switching oligonucleotides and to monitor their phenotype and proliferative and suppressive activity. We demonstrated that Tregs from peripheral blood from patients with multiple sclerosis preferentially expressed truncated splice variants, while the FL variant was the major variant in healthy donors. Tregs with induced expression of truncated FoxP3 splice variants demonstrated lower suppressive activity than those expressing FL variants. Reduced suppression was associated with the decreased expression of Treg-associated suppressive surface molecules and the production of cytokines. The deletion of exons 2 and/or 7 also reduced the cell proliferation rate. The results of this study show an association between FoxP3 splice variants and Treg function and proliferation. The modulation of Treg suppressive activity by the induction of the FoxP3 FL variant can become a promising strategy for regenerative immunotherapy.

Comparison of Alternative Splicing Landscapes Revealed by Long-Read Sequencing in Hepatocyte-Derived HepG2 and Huh7 Cultured Cells and Human Liver Tissue.

The long-read RNA sequencing developed by Oxford Nanopore Technologies provides a direct quantification of transcript isoforms, thereby making it possible to present alternative splicing (AS) profiles as arrays of single splice variants with different abundances. Additionally, AS profiles can be presented as arrays of genes characterized by the degree of alternative splicing (the DAS-the number of detected splice variants per gene). Here, we successfully utilized the DAS to reveal biological pathways influenced by the alterations in AS in human liver tissue and the hepatocyte-derived malignant cell lines HepG2 and Huh7, thus employing the mathematical algorithm of gene set enrichment analysis. Furthermore, analysis of the AS profiles as abundances of single splice variants by using the graded tissue specificity index τ provided the selection of the groups of genes expressing particular splice variants specifically in liver tissue, HepG2 cells, and Huh7 cells. The majority of these splice variants were translated into proteins products and appeal to be in focus regarding further insights into the mechanisms underlying cell malignization. The used metrics are intrinsically suitable for transcriptome-wide AS profiling using long-read sequencing.

The proper interplay between the expression of Spo11 splice isoforms and the structure of the pseudoautosomal region promotes XY chromosomes recombination

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11β splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11βki/− mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11βki/− males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11β and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.

Effective Human Motor Imagery Recognition via Segment Pool Based on One-Dimensional Convolutional Neural Network with Bidirectional Recurrent Attention Unit Network

Brain–computer interface (BCI) technology enables humans to interact with computers by collecting and decoding electroencephalogram (EEG) from the brain. For practical BCIs based on EEG, accurate recognition is crucial. However, existing methods often struggle to achieve a balance between accuracy and complexity. To overcome these challenges, we propose 1D convolutional neural networks with bidirectional recurrent attention unit network (1DCNN-BiRAU) based on a random segment recombination strategy (segment pool, SegPool). It has three main contributions. First, SegPool is proposed to increase training data diversity and reduce the impact of a single splicing method on model performance across different tasks. Second, it employs multiple 1D CNNs, including local and global models, to extract channel information with simplicity and efficiency. Third, BiRAU is introduced to learn temporal information and identify key features in time-series data, using forward–backward networks and an attention gate in the RAU. The experiments show that our model is effective and robust, achieving accuracy of 99.47% and 91.21% in binary classification at the individual and group levels, and 90.90% and 92.18% in four-category classification. Our model demonstrates promising results for recognizing human motor imagery and has the potential to be applied in practical scenarios such as brain–computer interfaces and neurological disorder diagnosis.

Splicing factors in the heart: Uncovering shared and unique targets

Alternative splicing generates specialized protein isoforms that allow the heart to adapt during development and disease. The recent discovery that mutations in the splicing factor RNA-binding protein 20 (RBM20) cause a severe form of familial dilated cardiomyopathy has sparked a great interest in alternative splicing in the field of cardiology. Since then, identification of splicing factors controlling alternative splicing in the heart has grown at a rapid pace. Despite the intriguing observation that a certain overlap exists between the targets of some splicing factors, an integrated and systematic analysis of their splicing networks is missing. Here, we compared the splicing networks of individual splicing factors by re-analyzing original RNA-sequencing data from eight previously published mouse models, in which a single splicing factor has been genetically deleted (i.e. HNRNPU, MBNL1/2, QKI, RBM20, RBM24, RBPMS, SRSF3, SRSF4). We show that key splicing events in Camk2d, Ryr2, Tpm1, Tpm2 and Pdlim5 require the combined action of the majority of these splicing factors. Additionally, we identified common targets and pathways among splicing factors, with the largest overlap between the splicing networks of MBNL, QKI and RBM24. We also re-analyzed a large-scale RNA-sequencing study on hearts of 128 heart failure patients. Here, we observed that MBNL1, QKI and RBM24 expression varied greatly. This variation in expression correlated with differential splicing of their downstream targets as found in mice, suggesting that aberrant splicing by MBNL1, QKI and RBM24 might contribute to the disease mechanism in heart failure.

Freddie: annotation-independent detection and discovery of transcriptomic alternative splicing isoforms using long-read sequencing.

Alternative splicing (AS) is an important mechanism in the development of many cancers, as novel or aberrant AS patterns play an important role as an independent onco-driver. In addition, cancer-specific AS is potentially an effective target of personalized cancer therapeutics. However, detecting AS events remains a challenging task, especially if these AS events are novel. This is exacerbated by the fact that existing transcriptome annotation databases are far from being comprehensive, especially with regard to cancer-specific AS. Additionally, traditional sequencing technologies are severely limited by the short length of the generated reads, which rarely spans more than a single splice junction site. Given these challenges, transcriptomic long-read (LR) sequencing presents a promising potential for the detection and discovery of AS. We present Freddie, a computational annotation-independent isoform discovery and detection tool. Freddie takes as input transcriptomic LR sequencing of a sample alongside its genomic split alignment and computes a set of isoforms for the given sample. It then partitions the input reads into sets that can be processed independently and in parallel. For each partition, Freddie segments the genomic alignment of the reads into canonical exon segments. The goal of this segmentation is to be able to represent any potential isoform as a subset of these canonical exons. This segmentation is formulated as an optimization problem and is solved with a dynamic programming algorithm. Then, Freddie reconstructs the isoforms by jointly clustering and error-correcting the reads using the canonical segmentation as a succinct representation. The clustering and error-correcting step is formulated as an optimization problem-the Minimum Error Clustering into Isoforms (MErCi) problem-and is solved using integer linear programming (ILP). We compare the performance of Freddie on simulated datasets with other isoform detection tools with varying dependence on annotation databases. We show that Freddie outperforms the other tools in its accuracy, including those given the complete ground truth annotation. We also run Freddie on a transcriptomic LR dataset generated in-house from a prostate cancer cell line with a matched short-read RNA-seq dataset. Freddie results in isoforms with a higher short-read cross-validation rate than the other tested tools. Freddie is open source and available at https://github.com/vpc-ccg/freddie/.

Proteogenomic Analysis of SF3B1mut CLL Reveals Altered Protein Signaling Cascades and Metabolomic Pathways

Although mutation in the U2 snRNP spliceosome component SF3B1 has been appreciated as a common driver of chronic lymphocytic leukemia (CLL) for over a decade, we still know little of its mechanism of action. Prior transcriptome-based characterizations have demonstrated widespread changes in alternative splicing in SF3B1mut CLL. We hypothesized that protein level characterization could potentially yield fresh insights on alternative expression of proteins and on the impact of altered signaling pathways in patients bearing this mutation. To this end, we assembled CLL specimens from a cohort of 105 patients enrolled on the German CLL Study Group (GCLLSG) trials CLL8 and CLL11, collected prior to therapy. Of these, 52 patients expressed SF3B1wt and 53 patients harbored clonal SF3B1mut (CCF>0.8); 25 of these were the K700E mutation. Additionally, we characterized these samples by WES (available for 97 patients) and RNA-Seq (available for 96 patients). Proteomes and phosphoproteomes from all 105 samples were analyzed using a multiplexed isobaric-tagged (TMT) high resolution mass spectrometry (MS) workflow. Briefly, cells were lysed and proteins were digested into peptides with LysC and trypsin. Protein yield per sample ranged from 20 μg to 300 μg. Peptides were labeled with TMT-11 reagent and combined into 11 multiplexes while accounting for input material availability and randomizing for SF3B1 mutation, IGHV status and ATM/TP53 mutation. To enable cross TMT-plex comparison, we constructed a common reference consisting of 40 samples with highest protein amount and an equal representation between SF3B1wt and SF3B1mut samples. Each plex was fractionated into 24 fractions by high pH reversed phase separation and 500 ng protein per fraction were analyzed by MS (Orbitrap Exploris, Thermo Fisher Scientific). For phosphoproteome analysis, peptides were pooled into 12 fractions and enriched by IMAC prior to MS analysis. Spectra were analyzed by Spectrum Mill v7.1 (Broad Institute) and searched against a human reference proteome database supplemented with non-canonical ORFs supported by Riboseq evidence of translation (nuORFdb v1.0). Patient specific proteins containing single amino variants, indels, and splice isoforms, derived from WES and RNA-Seq, were included in the search database. We obtained a total of 10,419 proteins, with a median of 8,882 proteins per TMT-plex and 6,969 proteins that were quantifiable across all samples. The phosphoproteome analysis identified 32,410 sites; while each TMT-plex yielded a median of 19,380 phosphosites per plex, 5,810 were quantifiable across all patients. We integrated proteome data with patient specific mutation calls, RNA-Seq and copy number alteration (CNA) data using Panoply, a cloud-based platform for proteogenomic data analysis (Broad Institute). We confirmed the impact of chr11 and chr13 deletion as well as chr12 amplification on protein expression. We readily identified peptides spanning the SF3B1mut amino acid regions in corresponding samples. By unsupervised clustering, samples were grouped by SF3B1 status on the CNA and proteome level, a trend not observed based on RNA or phosphoproteome analysis. Gene set enrichment analysis revealed enrichment of TNFA and PI3K signaling in the SF3B1wt clusters, while the SF3B1mut associated clusters showed a strong association with metabolism changes. This analysis was highly concordant with a parallel interrogation comparing proteins expressed in SF3B1wt CLL versus SF3B1mut CLL. Here 1,955 proteins and 1,737 phosphosites were significantly altered (5% FDR). Proteins involved in B cell receptor and MAP kinase signaling were highly expressed in SF3B1wt samples, while again amino acid metabolism pathways as well as oxidative phosphorylation were upregulated in SF3B1mut samples. To more deeply investigate the role of metabolism pathways in SF3B1mut CLL, we subjected 6 independent CLL samples to MS-based metabolomics analysis. Inosine (P=0.029) and guanosine (P=0.047) were lower in the SF3B1mut extracts (P=0.001), while acylcarnitines (P=0.0004) were increased, indicating fatty acid oxidation as a preferred energy source in SF3B1mut CLL. Altogether, our study reveals fresh evidence of altered energy metabolism in SF3B1mut CLL. Ongoing studies focus on defining the mechanism by which SF3B1mut exerts these metabolic changes and novel therapeutic opportunities afforded by these findings.

Whole exome sequencing in Brugada and long QT syndromes revealed novel rare and potential pathogenic mutations related to the dysfunction of the cardiac sodium channel

BackgroundBrugada syndrome (Brs) and long QT syndrome (LQTs) are the most observed “inherited primary arrhythmia syndromes” and “channelopathies”, which lead to sudden cardiac death.MethodsDetailed clinical information of Brs and LQTs patients was collected. Genomic DNA samples of peripheral blood were conducted for whole-exome sequencing on the Illumina HiSeq 2000 platform. Then, we performed bioinformatics analysis for 200 genes susceptible to arrhythmias and cardiomyopathies. Protein interaction and transcriptomic co-expression were analyzed using the online website and GTEx database.ResultsAll sixteen cases of Brs and six cases of LQTs were enrolled in the current study. Four Brs carried known pathogenic or likely pathogenic of single-point mutations, including SCN5A p.R661W, SCN5A p.R965C, and KCNH2 p.R692Q. One Brs carried the heterozygous compound mutations of DSG2 p.F531C and SCN5A p.A1374S. Two Brs carried the novel heterozygous truncated mutations (MAF < 0.001) of NEBL (p.R882X) and NPPA (p.R107X), respectively. Except for the indirect interaction between NEBL and SCN5A, NPPA directly interacts with SCN5A. These gene expressions had a specific and significant positive correlation in myocardial tissue, with high degrees of co-expression and synergy. Two Brs carried MYH7 p.E1902Q and MYH6 p.R1820Q, which were predicted as "damaging/possibly damaging" and "damaging/damaging" by Polyphen and SIFT algorithm. Two LQTs elicited the pathogenic single splicing mutation of KCNQ1 (c.922-1G > C). Three LQTs carried a single pathogenic mutation of SCN5A p.R1880H, KCNH2 p.D161N, and KCNQ1 p.R243S, respectively. One patient of LQTs carried a frameshift mutation of KCNH2 p. A188Gfs*143.ConclusionsThe truncated mutations of NEBL (p.R882X) and NPPA (p.R107X) may induce Brugada syndrome by abnormally affecting cardiac sodium channel. SCN5A (p.R661W, p.R965C and p.A1374S) and KCNH2 (p.R692Q) may cause Brugada syndrome, while SCN5A (p.R1880H), KCNQ1 (c.922-1G > C and p.R243S) and KCNH2 (p.D161N and p.A188Gfs*143) may lead to long QT syndrome.

Allele-specific alternative splicing of Drosophila Ribosomal protein S21 suppresses a lethal mutation in the Phosphorylated adaptor for RNA export (Phax) gene.

Genetic disruptions to the biogenesis of spliceosomal small-nuclear ribonucleoproteins in Drosophila cause wide-spread alternative splicing changes, including changes to the splicing of pre-mRNA for Ribosomal protein S21 (RpS21). Using a transposon mutant for the Phosphorylated adaptor for RNA export (Phax) gene, we demonstrate that changes in the splicing of RpS21 transcripts have a strong influence on the developmental progression of PhaxSH/SH mutants. Different alleles of the Drosophila RpS21 gene are circulating in common laboratory strains and cell lines. These alleles exhibit differences in RpS21 intron retention and splicing efficiency. Differences in the splicing of RpS21 transcripts account for prior conflicting observations of the phenotypic severity of PhaxSH/SH mutant stocks. The alleles uncover a strong splicing enhancer in RpS21 transcripts that can fully suppress the larval lethality and partially suppress the pupal lethality exhibited by PhaxSH/SH mutant lines. In the absence of the splicing enhancer, the splicing of RpS21 transcripts can be modulated in trans by the SR-rich B52 splicing factor. As PhaxSH/SH mutants exhibit wide-spread splicing changes in transcripts for other genes, findings here establish the importance of a single alternative splicing event, RpS21 splicing or intron retention, to the developmental progression of Drosophila.

Research on Modeling of Milling Force of Ball-End Mill Cutter in Multi-hardness Splicing Area of Automobile Panel Die Steel

The structure and the cavity surface hardened steel mold of automobile cover is very complex. In the stamping process, the local structure bears higher extrusion stress than the type surface, which often causes local wear and even cracking. The process of multi hardness splicing joint is applied to ensure the service life of the die according to the features of each part of the mold. The impact load caused by the repeatedly cut-in which always leads to the instability of cutting force in the whole process. The machining quality of the mold surface is reduced. Aiming at the machining process of the ball-end milling cutter in the mold splicing area, the impact at the moment of contact between the cutter and the workpiece is analyzed. The instantaneous chip thickness model that considers impact factors and the state model that calibrates the cutting state by coefficients is established. Finally, the prediction model of the milling force of the splicing area is obtained. The model is verified based on the machining cutting force measurement experiment in the case of die hardened steel Cr12MoV with single hardness and multi-hardness splicing. The average prediction error of experiments and simulation results is within 10%, which proves the accuracy of the model. The results show that the model has certain guiding significance for the actual processing of the splicing area of automobile mold cover parts.

Compressive performance of circular hollow section brace with eccentrically installed rib-stiffened splice plates

Hollow section braces are often adopted in steel frame structures because of their superior performance. To connect tubular members with framing elements, field-bolted connections of the slotted-in single splice plate and gusset plate are majorly used because of their ease of construction. However, the eccentricity between the splice and gusset plate axes reduces the compressive strength of the brace and induces local connection failures under seismic excitations. To improve the compressive strength, this paper proposes a new connection configuration for circular hollow section braces in which cruciform rib-stiffened splice plates are eccentrically installed such that the gusset plate axis coincides with the brace axis. The concept of the proposed connection was first described with theoretical considerations. To demonstrate the effectiveness of the proposed connections, compressive loading tests and subsequent cyclic loading tests were conducted for four test specimens. Furthermore, finite element analyses with long-brace models were performed to demonstrate the good performance of the proposed connection for a wide range of slenderness ratios.

Muscle stem cell polarity requires QKI-mediated alternative splicing of Integrin Alpha-7 (Itga7).

Muscle stem cells (MuSCs) have the ability to carry out the specialized function of cell polarization, which is required for the production of one repopulating cell and one myogenic progenitor cell with muscle regeneration capabilities. The mechanisms which regulate proteins involved in establishing MuSC polarity such as Dmd and Itga7 are currently not well understood. Herein, we define the RNA-binding protein Quaking (QKI) as a major regulator alternative splicing of key MuSC polarity factors including Dmd, Itga7, Mark2, and Numb. We generate a conditional QKI knockout mouse, and for the first time it is shown in vivo that deficiency of QKI in MuSCs results in reduced asymmetric cell divisions, leading to a loss of the myogenic progenitor cell population and striking muscle regeneration defects. Transcriptomic analysis of QKI-deficient MuSCs identifies QKI as a regulator of the splicing events which give rise to the mutually exclusive Itga7-X1 and -X2 isoforms. We observe increased X1 expression in QKI-deficient MuSCs and recapitulate this splicing event using antisense oligonucleotide directed against a quaking binding site within the Itga7 mRNA. Interestingly, recreating this single splicing event is detrimental to the polarization of Itga7 and Dmd proteins, and leads to a drastic reduction of the myogenic progenitor population, highlighting the significance of QKI-mediated alternative splicing of Itga7 in maintaining MuSC polarity. Altogether, these findings define a novel role for QKI as a post-transcriptional regulator of MuSC polarity.

N-cutting site of DNA splicing language for single string and palindromic rule

A new symbolization of Yusof-Goode (Y-G) splicing system was introduced by Yusof in 2012, is inspired by the framework of Formal Language Theory introduced by Head in 1987. Y-G splicing system is intended to present the biological process of DNA splicing in a translucent way. In this paper, starting with some relevant preliminaries, one theorem is proposed via Y-G approach using one initial string and one rule with different characteristics of the restriction enzyme. Additionally, the theorem showed the behavior of the splicing languages generated at single stage splicing. Two cases are considered in the theorem by conducting splicing using palindromic rule and palindromic recognition site with same left and right context for Case I and different left and right context for Case II. Furthermore, two molecular examples are discussed to validate two cases proposed in the theorem, which shows the real meaning of the theorem in biological aspect. From the proposed theorem, based on the splicing language generated, the type of splicing language can be determined. It is discovered that, the generated languages are in the form of limit and transient.

Simultaneous measurement of gas pressure and temperature based on Fabry–Pérot cavity cascading fiber Bragg grating

A novel optical fiber sensor with the ability to simultaneously measure gas pressure and temperature is proposed and experimentally demonstrated. The sensor comprises a fiber Bragg grating (FBG) and Fabry–Pérot interferometer (FPI) cascading; the FPI consists of two sections of single mode fiber splicing a short quartz capillary with a gas channel prepared by femtosecond laser micromachining that circulates the Fabry–Pérot cavity with external gas. Because the open cavity FPI is only sensitive to gas pressure and extremely less sensitive to temperature, while FBG is only sensitive to temperature and insensitive to gas pressure, an extremely simple diagonal matrix of sensitivity is obtained. The diagonal matrix elements are temperature and gas pressure sensitivity, corresponding to 0.012 nm/°C and 4.14 nm/MPa, respectively. Experimental results show that the sensor structure can realize the simultaneous measurement of gas pressure and temperature and eliminate the cross sensitivity between the gas pressure and temperature. The proposed sensor has the advantages of compact structure, simple fabrication, and convenient packaging and has a wide application prospect in the field of simultaneous measurement of gas pressure and temperature.

Human APOBEC3 Variations and Viral Infection

Human APOBEC3 (apolipoprotein B mRNA-editing catalytic polypeptide-like 3) enzymes are capable of inhibiting a wide range of endogenous and exogenous viruses using deaminase and deaminase-independent mechanisms. These enzymes are essential components of our innate immune system, as evidenced by (a) their strong positive selection and expansion in primates, (b) the evolution of viral counter-defense mechanisms, such as proteasomal degradation mediated by HIV Vif, and (c) hypermutation and inactivation of a large number of integrated HIV-1 proviruses. Numerous APOBEC3 single nucleotide polymorphisms, haplotypes, and splice variants have been identified in humans. Several of these variants have been reported to be associated with differential antiviral immunity. This review focuses on the current knowledge in the field about these natural variations and their roles in infectious diseases.

Detection of Gene Mutations in Patients with Clinically Suspected Heritary Spherocytosis By Next Generation Sequencing

Introduction: Hereditary spherocytosis (HS) is the most frequent form of inherited hemolytic anemia characterized by the presence of spherical-shaped erythrocytes on the peripheral blood smear, hemolysis, splenomegaly, jaundice, and gallstones. The clinical severity of HS varied widely, and its prevalence is 1 in 2,000 in Northern European population and1 in 100,000 in China. The diagnosis can be based on the physical examination, complete red blood cell count, reticulocytes count, medical history and specific tests, preferentially the EMA test test and AGLT, as well as molecular diagnosis particularly gene sequencing. To date, mutations in at least five genes (ANK1, EPB42, SLC4A1, SPTA1, and SPTB) are associated with different subtypes of HS. Next-generation gene sequencing (NGS) is a high-throughput parallel sequencing method and its high-sensitivity is widely used in variation identification in clinical settings with a wide range of human diseases including inherited disorders. This study is aim to discover pathogenic mutations in clinically suspected HS patients by NGS to confirm and/or discriminate diagnosis.Methods: A cohort of seven patients with clinically suspected HS were enrolled in this study. The DNA samples were extracted from peripheral blood samples of the patients and their parents (if available), and were sheared into 200-300bp fragments using the Covaris ultrasonic instrument, followed by whole-exome sequencing. NGS samples were sequenced on the Illumina HiSeq X10 instrument according to manufacturer's protocol. Raw data analysis was performed with the Illumina Pipeline software and the identified mutations were confirmed by Sanger sequencing. Sequences were aligned to the hg19 human genome reference using information from databases including NCBI dbSNP, HapMap, and the 1000 Human Genomes Project dataset. SOAPsnp and Samtools were used to locate the mutations, and the potential functional impacts of the identified variants were assessed using SIFT, PolyPhen-2 and MutationTaster software.Results: Seven patients with age from 8-44yrs were enrolled in our cohort study. NGS results have identified 4 missense mutation SLC4A1 (P854L), SLC4A1 (P455R), SEC23B (P25H and P567R), 3 nonsense mutation ANK1 (R1334X), SPTB (R1756X), SPTB (E1602X), and 1 splicing mutation SPTB (c.5937+1G>C) in seven patients respectively. Three novel mutations, SPTB (R1756X and E1602X) that were stop-gain single nucleotide variants (SNVs) and splicing variant SPTB (c.5937+1G>C) were identified responsible for hereditaryspherocytosis (HS). Considering that the missense mutation SLC4A1 (P854L) was inherited from asymptomatic mother, the pathogenicity of this site is low. Moreover, the mutation SPTB (E1602X) is a de novo mutation and thus likely to be pathogenic. However, the compound heterozygous mutation SEC23B (P25H and P567R) is responsible for congenital dyserythropoietic anemia (CDA) which represent similar clinical symptoms with HS, may have been misdiagnosed and needs to be further studied.Conclusions: Together, we have identified three novel mutations, two stop-gain mutation SPTB (R1756X and E1602X) and one splicing variant SPTB (c.5937+1G>C), in hereditary spherocytosis patients. Our results suggested that genetic diagnosis by NGS is a fast, economic and accurate way to detect and identify pathogenic alterations of inherited diseases like hereditary spherocytosis, and more importantly a useful tool for differential diagnosis. DisclosuresNo relevant conflicts of interest to declare.

An identical-by-descent novel splice-donor variant in PRUNE1 causes a neurodevelopmental syndrome with prominent dystonia in two consanguineous Sudanese families.

PRUNE1 is linked to a wide range of neurodevelopmental and neurodegenerative phenotypes. Multiple pathogenic missense and stop-gain PRUNE1 variants were identified in its DHH and DHHA2 phosphodiesterase domains. Conversely, a single splice alteration was previously reported. We investigated five patients from two unrelated consanguineous Sudanese families with an inherited severe neurodevelopmental disorder using whole-exome sequencing coupled with homozygosity mapping, segregation, and haplotype analysis. We identified a founder haplotype transmitting a homozygous canonical splice-donor variant (NM_021222.3:c.132+2T>C) in intron 2 of PRUNE1 segregated with the phenotype in all the patients. This splice variant possibly results in an in-frame deletion in the DHH domain or premature truncation of the protein. The phenotypes of the affected individuals showed phenotypic similarities characterized by remarkable pyramidal dysfunction and prominent extrapyramidal features (severe dystonia and bradykinesia). In conclusion, we identified a novel founder variant in PRUNE1 and corroborated abnormal splicing events as a disease mechanism in PRUNE1-related disorders. Given the phenotypes' consistency coupled with the founder effect, canonical and cryptic PRUNE1 splice-site variants should be carefully evaluated in patients presenting with prominent dystonia and pyramidal dysfunction.

Transcriptomic analysis of the trade-off between endurance and burst-performance in the frog Xenopus allofraseri

BackgroundVariation in locomotor capacity among animals often reflects adaptations to different environments. Despite evidence that physical performance is heritable, the molecular basis of locomotor performance and performance trade-offs remains poorly understood. In this study we identify the genes, signaling pathways, and regulatory processes possibly responsible for the trade-off between burst performance and endurance observed in Xenopus allofraseri, using a transcriptomic approach.ResultsWe obtained a total of about 121 million paired-end reads from Illumina RNA sequencing and analyzed 218,541 transcripts obtained from a de novo assembly. We identified 109 transcripts with a significant differential expression between endurant and burst performant individuals (FDR ≤ 0.05 and logFC ≥2), and blast searches resulted in 103 protein-coding genes. We found major differences between endurant and burst-performant individuals in the expression of genes involved in the polymerization and ATPase activity of actin filaments, cellular trafficking, proteoglycans and extracellular proteins secreted, lipid metabolism, mitochondrial activity and regulators of signaling cascades. Remarkably, we revealed transcript isoforms of key genes with functions in metabolism, apoptosis, nuclear export and as a transcriptional corepressor, expressed in either burst-performant or endurant individuals. Lastly, we find two up-regulated transcripts in burst-performant individuals that correspond to the expression of myosin-binding protein C fast-type (mybpc2). This suggests the presence of mybpc2 homoeologs and may have been favored by selection to permit fast and powerful locomotion.ConclusionThese results suggest that the differential expression of genes belonging to the pathways of calcium signaling, endoplasmic reticulum stress responses and striated muscle contraction, in addition to the use of alternative splicing and effectors of cellular activity underlie locomotor performance trade-offs. Ultimately, our transcriptomic analysis offers new perspectives for future analyses of the role of single nucleotide variants, homoeology and alternative splicing in the evolution of locomotor performance trade-offs.