Identification of alternative splicing regulatory patterns and characteristic splicing factors in heart failure using RNA-seq data and machine learning.

Heart failure (HF) represents the advanced stage of several cardiovascular disorders. This study aimed to build an alternative splicing regulatory network and identify potential splicing factors involved in HF utilizing RNA-seq data and machine learning algorithms. We performed bioinformatics analysis on RNA-seq datasets containing samples from HF patients and normal individuals to obtain gene expression matrices and identify differently regulated alternative splicing events in HF. By calculating percent spliced-in (PSI) value, we identified 4055 abnormal alternative splicing events of 3142 genes in HF. These genes were significantly enriched in PPAR signaling, regulation of actin cytoskeleton, and muscle contraction. Interestingly, based on abnormal alternative splicing events, two distinct clusters of HF patients with distinct molecular mechanisms and pathways were identified using unsupervised clustering. Additionally, we built a regulatory network consisting of heart failure-related alternative splicing and splicing factors. Subsequently, we identify 203 HF specific pairs between splicing factors and alternative splicing events. Four splicing factors (RBM5, ZRANB2, HnRNPF, and HnRNPA0) were found using LASSO and SVM-RFE algorithms, their expression patterns were confirmed in two other microarray datasets. Our study clarifies involvement of splicing factors and alternative splicing events in HF by thoroughly analyzing RNA-seq data with machine learning methods. The findings may advance our understanding of the regulatory systems underlying biological processes associated with heart failure by providing candidates for further investigation and markers for diagnostic and therapeutic purposes.

Introns with branchpoint-distant 3' splice sites: Splicing mechanism and regulatory roles.

The two transesterification reactions of pre-mRNA splicing require highly complex yet well-controlled rearrangements of small nuclear RNAs and proteins (snRNP) in the spliceosome. The efficiency and accuracy of these reactions are critical for gene expression, as almost all human genes pass through pre-mRNA splicing. Key parameters that determine the splicing outcome are the length of the intron, the strengths of its splicing signals and gaps between them, and the presence of splicing controlling elements. In particular, the gap between the branchpoint (BP) and the 3' splice site (ss) of introns is a major determinant of the splicing efficiency. This distance falls within a small range across the introns of an organism. The constraints exist possibly because BP and 3'ss are recognized by BP-binding proteins, U2 snRNP and U2 accessory factors (U2AF) in a coordinated manner. Furthermore, varying distances between the two signals may also affect the second transesterification reaction since the intervening RNA needs to be accurately positioned within the complex RNP machinery. Splicing such pre-mRNAs requires cis-acting elements in the RNA and many trans-acting splicing regulators. Regulated pre-mRNA splicing with BP-distant 3'ss adds another layer of control to gene expression and promotes alternative splicing.

The role of BUD31 in clear cell renal cell carcinoma: prognostic significance, alternative splicing, and tumor immune environment.

BUD31, a splicing factor, is linked to various cancers. This study examines BUD31's expression, prognostic value, mutation profile, genomic instability, tumor immune environment, and role in clear cell renal cell carcinoma (ccRCC), focusing on cell cycle regulation via alternative splicing. BUD31 expression was analyzed using TCGA and GTEx databases across 33 cancers. Techniques included IHC staining, survival analysis, Cox regression, and nomogram construction. Mutation landscape, genomic instability, and tumor immune microenvironment were evaluated. Functional assays on ccRCC cell lines involved BUD31 knockdown, RNA sequencing, and alternative splicing analysis. BUD31 was upregulated in multiple tumors, including ccRCC. High BUD31 expression correlated with worse survival outcomes and was identified as an independent predictor of poor prognosis in ccRCC. High BUD31 expression also correlated with increased genomic instability and a less active immune microenvironment. BUD31 knockdown inhibited cell proliferation, migration, and invasion in vitro and reduced tumor growth in vivo. RNA sequencing identified 390 alternative splicing events regulated by BUD31, including 17 cell cycle-related genes. KEGG analysis highlighted pathways involved in cell cycle regulation, indicating BUD31's role in promoting cell cycle progression through alternative splicing. BUD31 is upregulated in various tumors and is associated with poor outcomes, increased genomic instability, and a suppressed immune microenvironment in ccRCC. BUD31 promotes cell cycle progression via alternative splicing, suggesting it as a prognostic biomarker and potential therapeutic target in ccRCC.

Identification of a prognostic gene signature in patients with cisplatin resistant squamous cell lung cancer.

Background: In the absence of targeted mutations and immune checkpoints, platinum-based chemotherapy remains a gold standard agent in the treatment of patients with lung squamous cell carcinoma (LUSC). However, cisplatin resistance greatly limits its therapeutic efficacy and presents challenges in the treatment of lung cancer patients. Therefore, the potential clinical needs for this research focus on identifying novel molecular signatures to further elucidate the underlying mechanisms of cisplatin resistance in LUSC. A growing body of evidence indicates that alternative splicing (AS) events significantly influence the tumor progression and survival of patients with LUSC. However, there are few systematic analyses of AS reported in LUSC. This study aims to explore the role of messenger RNA (mRNA), microRNA (miRNA), and AS in predicting prognosis in patients with cisplatin-resistant LUSC and provide potential therapeutic targets and drugs. Methods: Gene expression and miRNA expression, using RNA sequencing (RNA-seq), and SpliceSeq data were downloaded from The Cancer Genome Atlas (TCGA) database. The least absolute shrinkage and selection operator (LASSO) Cox regression analysis were used to construct predictive models. Kaplan-Meier survival analyses were used to evaluate patients' prognosis. Single-sample gene set enrichment analysis (ssGSEA) conducted via the R package "GSEAbase" was used to evaluate the immune-related characteristics. Immunohistochemistry was used to examine protein expression. The Connectivity Map (CMap) database was used to screen for potential drugs. The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was used to determine and calculate the half-maximal inhibitory concentration (IC50) of the drugs, sulforaphane and parthenolide. Results: In this study, bioinformatics were used to identify mRNAs, miRNAs, and AS events related to response to cisplatin and to establish an integrated prognostic signature for 70 patients with LUSC and cisplatin resistance. The prognostic signature served as an independent prognostic factor with high accuracy [hazard ratio (HR) =2.346, 95% confidence interval (CI): 1.568-3.510; P<0.001], yielding an area under the curve (AUC) of 0.825, 0.829, and 0.877 for 1-, 3-, and 5-year survival, respectively. It also demonstrated high predictive performance in this cohort of patients with LUSC, with an AUC of 0.734, 0.767, and 0.776 for 1-, 3-, and 5-year survival, respectively. This integrated signature was also found to be an independent indicator among conventional clinical features (HR =2.288, 95% CI: 1.547-3.383; P<0.001). In addition, we analyzed the correlation of the signature with immune infiltration and identified several small-molecule drugs that had the potential to improve the survival of patients with LUSC. Conclusions: This study provides a framework for the mRNA-, miRNA-, and AS-based evaluation of cisplatin response and several potential therapeutic drugs for targeting cisplatin resistance in LUSC. These findings may serve as a theoretical basis for the clinical alleviation of cisplatin resistance and thus help to improve treatment responses to chemotherapy in patients with LUSC.

Molecular impact of mutations in RNA splicing factors in cancer.

Somatic mutations in genes encoding components of the RNA splicing machinery occur frequently in multiple forms of cancer. The most frequently mutated RNA splicing factors in cancer impact intronic branch site and 3' splice site recognition. These include mutations in the core RNA splicing factor SF3B1 as well as mutations in the U2AF1/2 heterodimeric complex, which recruits the SF3b complex to the 3' splice site. Additionally, mutations in splicing regulatory proteins SRSF2 and RBM10 are frequent in cancer, and there has been a recent suggestion that variant forms of small nuclear RNAs (snRNAs) may contribute to splicing dysregulation in cancer. Here, we describe molecular mechanisms by which mutations in these factors alter splice site recognition and how studies of this process have yielded new insights into cancer pathogenesis and the molecular regulation of splicing. We also discuss data linking mutant RNA splicing factors to RNA metabolism beyond splicing.

Unraveling novel mRNA transcripts of the human DNA N-glycosylase 1 (NTHL1) gene with the implementation of an innovative targeted DNA-seq assay.

The human NTHL1 gene encodes a DNA glycosylase that plays a key role in the base excision repair (BER) pathway, repairing oxidative DNA damage and maintaining genome integrity. The physiological activity of NTHL1 is crucial in preventing genetic alterations that can lead to cancer. In this study, we employed an innovative targeted DNA sequencing (DNA-seq) methodology to explore the transcriptional landscape of the NTHL1 gene, revealing previously uncharacterized alternative splicing events and novel exons. Our designed approach provided significantly improved sequencing depth and coverage, enabling the identification of novel NTHL1 mRNA transcripts. Bioinformatics analysis confirmed all annotated splice junctions of the main NTHL1 transcripts (v.1 - v.3) and revealed novel mRNA transcripts (NTHL1 v.4 - v.9) derived from splicing events between annotated exons as well as mRNAs containing previously uncharacterized exons (NTHL1 v.10 - v.14). Quantitative PCR analysis highlighted a diverse expression pattern of these novel transcripts across different human cell lines, suggesting cell-specific roles and regulatory mechanisms. Notably, NTHL1 v.5 was overexpressed in luminal A breast cancer cells (MCF-7), while v.13 was prominent in triple negative (BT-20), HER2 + breast cancer (SK-BR-3), prostate, colorectal cancer cells and HEK-293 cells. Our findings suggest that specific novel NTHL1 transcripts may encode protein isoforms with distinct structural features, as indicated by ribosome profiling datasets, while others containing premature termination codons could function as long non-coding RNAs. These insights enhance our understanding of NTHL1 regulatory role and its potential as a biomarker and therapeutic target in human malignancies. This study underscores the importance of exploring the transcriptional diversity of NTHL1 to fully elucidate its role in cancer pathobiology.

Genome-wide identification of pan-cancer common and cancer-specific alternative splicing events in 9 types of cancer.

Alternative splicing (AS) has significant clinical relevance with cancers and is a potential source of neoepitopes. In this study, RNA-seq data of 94 solid tumor and matched adjacent normal tissues from 47 clinical patients covering nine cancer types were comprehensively analyzed using SUVA developed by ourselves. The results identified highly conserved pan-cancer differential alternative splicing (DAS) events and cancer-specific DAS events in a series of tumor samples, which in turn revealed the heterogeneity of AS post-transcriptional regulation across different cancers. The co-disturbed network between spliceosome factors (SFs) and common cancer-associated DAS was further constructed, suggesting the potential possibility of the regulation of differentially expressed SFs on DAS. Finally, the common cancer-associated DAS events were fully validated using the TCGA dataset, confirming the significant correlation between cancer-associated DAS and prognosis. Briefly, our study elucidates new insights into conservatived and specific DAS in cancer, providing valuable resources for cancer therapeutic targets.

LncRNA HOTAIRM1 promotes radioresistance in nasopharyngeal carcinoma by modulating FTO acetylation-dependent alternative splicing of CD44.

BACKGROUND: Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC); however, almost 20% of patients experience treatment failure due to radioresistance. Therefore, understanding the mechanisms of radioresistance is imperative. HOTAIRM1 is deregulated in various human cancers, yet its role in NPC radioresistance are largely unclear. METHODS: This study investigated the association between HOTAIRM1 and radioresistance using CCK8, flow cytometry, and comet assays. Additionally, xenograft mice and patient-derived xenografts (PDX) models were employed to elucidate the biological functions of HOTAIRM1, and transcriptomic RNA sequencing was utilized to identify its target genes. RESULTS: Our study revealed an upregulation of HOTAIRM1 levels in radioresistant NPC cell lines and tissues. Furthermore, a positive correlation was noted between high HOTAIRM1 expression and increased NPC cell proliferation, reduced apoptosis, G2/M cell cycle arrest, and diminished cellular DNA damage following radiotherapy. HOTAIRM1 modulates the acetylation and stability of the FTO protein, and inhibiting FTO elevates the m6A methylation level of CD44 precursor transcripts in NPC cells. Additionally, silencing the m6A reading protein YTHDC1 was found to increase the expression of CD44V. HOTAIRM1 enhances NPC cell resistance to ferroptosis and irradiation through the HOTAIRM1-FTO-YTHDC1-CD44 axis. Mechanistically, HOTAIRM1 interacts with the FTO protein and induces m6A demethylation of the CD44 transcript. The absence of m6A modification in the CD44 transcript prevents its recognition by YTHDC1, resulting in the transition from CD44S to CD44V. An abundance of CD44V suppresses ferroptosis induced by irradiation and contributes to NPC radioresistance. CONCLUSIONS: In conclusion, the results in this study support the idea that HOTAIRM1 stimulates CD44 alternative splicing via FTO-mediated demethylation, thereby attenuating ferroptosis induced by irradiation and promoting NPC radioresistance.

Splice modulation strategy applied to deep intronic variants in COL7A1 causing recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1. One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.

Dynamics and Evolutionary Conservation of B Complex Protein Recruitment During Spliceosome Activation.

Spliceosome assembly and catalytic site formation (called activation) involve dozens of protein and snRNA binding and unbinding events. The B-complex specific proteins Prp38, Snu23, and Spp381 have critical roles in stabilizing the spliceosome during conformational changes essential for activation. While these proteins are conserved, different mechanisms have been proposed for their recruitment to spliceosomes. To visualize recruitment directly, we used Colocalization Single Molecule Spectroscopy (CoSMoS) to study the dynamics of Prp38, Snu23, and Spp381 during splicing in real time. These proteins bind to and release from spliceosomes simultaneously and are likely associated with one another. We designate the complex of Prp38, Snu23, and Spp381 as the B Complex Protein (BCP) subcomplex. Under splicing conditions, the BCP associates with pre-mRNA after tri-snRNP binding. BCP release predominantly occurs after U4 snRNP dissociation and after NineTeen Complex (NTC) association. Under low concentrations of ATP, the BCP pre-associates with the tri-snRNP resulting in their simultaneous binding to pre-mRNA. Together, our results reveal that the BCP recruitment pathway to the spliceosome is conserved between S. cerevisiae and humans. Binding of the BCP to the tri-snRNP when ATP is limiting may result in formation of unproductive complexes that could be used to regulate splicing.

Productive mRNA Chromatin Escape is Promoted by PRMT5 Methylation of SNRPB.

Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A) -but not type I PRMT inhibition-leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.

A missense variant in SLC12A3 gene enhances aberrant splicing causing Gitelman syndrome.

Gitelman syndrome (GS) is the most prevalent genetic tubulopathy characterized by several electrolyte abnormalities, including hypokalemia, hypomagnesemia, hypocalciuria, metabolic alkalosis, and hyperreninemic hyperaldosteronism. These features are caused by a bi-allelic mutation in the SLC12A3 gene. In this report, we present a case of GS in an asymptomatic woman who incidentally exhibited hypokalemia during an antenatal check-up. Her biochemical profile was consistent with GS. Genetic analysis revealed two heterozygous variants in trans, namely, NM_001126108.2:c.625C>T; p.(Arg209Trp) and c.965C>T; p.(Ala322Val). The c.625C>T; p.(Arg209Trp) variant has previously been experimentally confirmed as a loss-of-function (LOF) variant. However, the functional impact of the c.965C>T variant, located at the 5 prime end of exon 8, has not been fully elucidated. Through the utilization of both complementary DNA (cDNA) and minigene analysis, we confirmed that the c.965C>T variant can generate two distinct cDNA transcripts. The first transcript carries a missense mutation, p.(Ala322Val) in the full SLC12A3 transcript, while the second transcript consists of an in-frame deletion of both exons 7 and 8 in the SLC25A13 transcript, in which may result in the loss of transmembrane regions 5 - 6 involved in chloride transport. Our findings provide insights into the intricate mechanisms of splicing, highlighting how a variant in one exon can remotely influence the transcription of an upstream exon, as observed with the variant in exon 8 impacting the transcription of exon 7.

Analysis of the complete mitochondrial genome of Panax quinquefolius reveals shifts from cis-splicing to trans-splicing of intron cox2i373.

Panax quinquefolius is a perennial plant with medicinal values. In this study, we assembled the complete mitochondrial genome (mitogenome) of P. quinquefolius using PMAT assembler. The total length of P. quinquefolius mitogenome is 573,154 bp. We annotated a total of 34 protein-coding genes (PCGs), 35 tRNA genes, and 6 rRNA genes in this mitogenome. The analysis of repetitive elements shows that there are 153 SSRs, 24 tandem repeats and 242 pairs of dispersed repeats this mitogenome. Also, we found 24 homologous sequences with a total length of 64,070 bp among its mitogenome and plastome, accounting for 41.05 % of the plastome, and 11.18 % of the mitogenome, showing a remarkable frequent sequence dialogue between plastome and mitogenomes. Besides, a total of 583 C to U RNA editing sites on 34 PCGs of high confidence were predicted by using Deepred-mt. We also inferred the phylogenetic relationships of P. quinquefolius and other angiosperms based on mitochondrial PCGs. Finally, we observed a shift from cis- to trans-splicing in P. quinquefolius for two mitochondrial introns, namely cox2i373 and nad1i728, and a pair of 48 bp short repetitive sequences may be associated with the breaking and rearrangement of the cox2i373 intron. The fragmentation of the cox2i373 intron was further confirmed by our PCR amplification experiments. In summary, our report on the P. quinquefolius mitogenome provides a new perspective on the intron evolution of the mitogenome.

Multi-faceted regulation of CREB family transcription factors.

cAMP response element-binding protein (CREB) is a ubiquitously expressed nuclear transcription factor, which can be constitutively activated regardless of external stimuli or be inducibly activated by external factors such as stressors, hormones, neurotransmitters, and growth factors. However, CREB controls diverse biological processes including cell growth, differentiation, proliferation, survival, apoptosis in a cell-type-specific manner. The diverse functions of CREB appear to be due to CREB-mediated differential gene expression that depends on cAMP response elements and multi-faceted regulation of CREB activity. Indeed, the transcriptional activity of CREB is controlled at several levels including alternative splicing, post-translational modification, dimerization, specific transcriptional co-activators, non-coding small RNAs, and epigenetic regulation. In this review, we present versatile regulatory modes of CREB family transcription factors and discuss their functional consequences.

A defective splicing machinery promotes senescence through MDM4 alternative splicing.

Defects in the splicing machinery are implicated in various diseases, including cancer. We observed a general reduction in the expression of spliceosome components and splicing regulators in human cell lines undergoing replicative, stress-induced, and telomere uncapping-induced senescence. Supporting the view that defective splicing contributes to senescence, splicing inhibitors herboxidiene, and pladienolide B induced senescence in normal and cancer cell lines. Furthermore, depleting individual spliceosome components also promoted senescence. All senescence types were associated with an alternative splicing transition from the MDM4-FL variant to MDM4-S. The MDM4 splicing shift was reproduced when splicing was inhibited, and spliceosome components were depleted. While decreasing the level of endogenous MDM4 promoted senescence and cell survival independently of the MDM4-S expression status, cell survival was also improved by increasing MDM4-S. Overall, our work establishes that splicing defects modulate the alternative splicing of MDM4 to promote senescence and cell survival.

Molecular cloning and functional characterization of mitochondrial RNA splicing 2 in fish Megalobrama amblycephala, and its potential roles in magnesium homeostasis and mitochondrial function.

Mitochondrial function can be regulated by ion channels. Mitochondrial RNA splicing 2 (Mrs2) is a magnesium ion (Mg2+) channel located in the inner mitochondrial membrane, thereby mediating the Mg2+ influx into the mitochondrial matrix. However, its potential role in regulating the Mg homeostasis and mitochondrial function in aquatic species is still unclear. This study molecularly characterizes the gene encoding Mrs2 in fish M. amblycephala with its functions in maintaining the Mg homeostasis and mitochondrial function verified. The mrs2 gene is 2133 bp long incorporating a 1269 bp open reading frame, which encodes 422 amino acids. The Mrs2 protein includes two transmembrane domains and a conserved tripeptide Gly-Met-Asn, and has a high homology (65.92-97.64%) with those of most vertebrates. The transcript of mrs2 was relatively high in the white muscle, liver and kidney. The inhibition of mrs2 reduces the expressions of Mg2+ influx/efflux-related proteins, mitochondrial Mg content, and the activities of mitochondrial complex I and V in hepatocytes. However, the over-expression of mrs2 increases the expressions of Mg2+ influx/efflux-related proteins, mitochondrial Mg content, and the complex V activity, but decreases the activities of mitochondrial complex III and IV and citrate synthase in hepatocytes. Collectively, Mrs2 is highly conserved among different species, and is prerequisite for maintaining Mg homeostasis and mitochondrial function in fish.

The cap-binding complex modulates ABA-responsive transcript splicing during germination in barley (Hordeum vulgare).

To decipher the molecular bases governing seed germination, this study presents the pivotal role of the cap-binding complex (CBC), comprising CBP20 and CBP80, in modulating the inhibitory effects of abscisic acid (ABA) in barley. Using both single and double barley mutants in genes encoding the CBC, we revealed that the double mutant hvcbp20.ab/hvcbp80.b displays ABA insensitivity, in stark contrast to the hypersensitivity observed in single mutants during germination. Our comprehensive transcriptome and metabolome analysis not only identified significant alterations in gene expression and splicing patterns but also underscored the regulatory nexus among CBC, ABA, and brassinosteroid (BR) signaling pathways.

Whole paternal uniparental disomy of chromosome 4 with a novel homozygous IDUA splicing variant, c.159-9T>A, in a Chinese patient with mucopolysaccharidosis type I.

BACKGROUND: Mucopolysaccharidosis type I (MPS-I) is a rare autosomal recessive genetic lysosomal storage disorder that is caused by pathogenic variants of the α-L-iduronidase (IDUA) gene. This study aimed to identify the genetic causes of MPS-I in a Chinese patient and construct a minigene of IDUA to analyze its variants upon splicing. METHODS: Whole-exome sequencing (WES) and Sanger sequencing were used to confirm the potential causative variants. Single-nucleotide polymorphism (SNP) array was subsequently performed to confirm uniparental disomy (UPD). Minigene assay was performed to analyze the effect on splicing of mRNA. We meanwhile explored the conservative analysis and protein homology simulation. RESULTS: A novel homozygous splicing mutation of IDUA, c.159-9T>A, was identified in an individual presenting with overlapping features of MPS-I. Interestingly, only the father and sisters, but not the mother, carried the variant in a heterozygous state. WES and SNP array analyses validated paternal UPD on chromosome 4. Minigene splicing revealed two aberrant splicing events: exon 2 skipping and intron 1 retention. Moreover, the specific structure of the mutant protein obviously changed according to the results of the homologous model. CONCLUSIONS: This study describes a rare autosomal recessive disorder with paternal UPD of chromosome 4 leading to the homozygosity of the IDUA splicing variant in patients with MPS-I for the first time. This study expands the variant spectrum of IDUA and provides insights into the splicing system, facilitating its enhanced diagnosis and treatment.

Splice-switching antisense oligonucleotides for pediatric neurological disorders.

Pediatric neurological disorders are frequently devastating and present unmet needs for effective medicine. The successful treatment of spinal muscular atrophy with splice-switching antisense oligonucleotides (SSO) indicates a feasible path to targeting neurological disorders by redirecting pre-mRNA splicing. One direct outcome is the development of SSOs to treat haploinsufficient disorders by targeting naturally occurring non-productive splice isoforms. The development of personalized SSO treatment further inspired the therapeutic exploration of rare diseases. This review will discuss the recent advances that utilize SSOs to treat pediatric neurological disorders.

Detecting image manipulation with ELA-CNN integration: a powerful framework for authenticity verification.

The exponential progress of image editing software has contributed to a rapid rise in the production of fake images. Consequently, various techniques and approaches have been developed to detect manipulated images. These methods aim to discern between genuine and altered images, effectively combating the proliferation of deceptive visual content. However, additional advancements are necessary to enhance their accuracy and precision. Therefore, this research proposes an image forgery algorithm that integrates error level analysis (ELA) and a convolutional neural network (CNN) to detect the manipulation. The system primarily focuses on detecting copy-move and splicing forgeries in images. The input image is fed to the ELA algorithm to identify regions within the image that have different compression levels. Afterward, the created ELA images are used as input to train the proposed CNN model. The CNN model is constructed from two consecutive convolution layers, followed by one max pooling layer and two dense layers. Two dropout layers are inserted between the layers to improve model generalization. The experiments are applied to the CASIA 2 dataset, and the simulation results show that the proposed algorithm demonstrates remarkable performance metrics, including a training accuracy of 99.05%, testing accuracy of 94.14%, precision of 94.1%, and recall of 94.07%. Notably, it outperforms state-of-the-art techniques in both accuracy and precision.