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High-throughput sequencing has identified numerous intronic variants in the SCN1A gene in epilepsy patients. Abnormal mRNA splicing caused by these variants can lead to significant phenotypic differences, but the mechanisms of epileptogenicity and phenotypic differences remain unknown. Two variants, c.4853-1 G>C and c.4853-25 T>A, were identified in intron 25 of SCN1A, which were associated with severe Dravet syndrome (DS) and mild focal epilepsy with febrile seizures plus (FEFS+), respectively. The impact of these variants on protein expression, electrophysiological properties of sodium channels and their correlation with epilepsy severity was investigated through plasmid construction and transfection based on the aberrant spliced mRNA. We found that the expression of truncated mutant proteins was significantly reduced on the cell membrane, and retained in the cytoplasmic endoplasmic reticulum. The mutants caused a decrease in current density, voltage sensitivity, and an increased vulnerability of channel, leading to a partial impairment of sodium channel function. Notably, the expression of DS-related mutant protein on the cell membrane was higher compared to that of FEFS+-related mutant, whereas the sodium channel function impairment caused by DS-related mutant was comparatively milder than that caused by FEFS+-related mutant. Our study suggests that differences in protein expression levels and altered electrophysiological properties of sodium channels play important roles in the manifestation of diverse epileptic phenotypes. The presence of intronic splice site variants may result in severe phenotypes due to the dominant-negative effects, whereas non-canonical splice site variants leading to haploinsufficiency could potentially cause milder phenotypes.

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BACKGROUND: Marfan syndrome (MFS) is a complex genetic systemic connective tissue disorder. It is well known that genetic factors play a critical role in the progression of MFS, with nearly all cases attributed to variants in the FBN1 gene. METHODS: We investigated a Chinese family with MFS spanning two generations. Whole exome sequencing, in silico analysis, minigene constructs, transfection, RT-PCR, and protein secondary structure analysis were used to analyze the genotype of the proband and his father. RESULTS: The main clinical manifestations of the proband and his father were subluxation of the left lens and high myopia with pectus deformity. Whole exome sequencing identified a novel single nucleotide variant (SNV) in the FBN1 gene at a non-canonical splice site, c.443-3C>G. This variant resulted in two abnormal mRNA transcripts, leading to a frameshift and an in-frame insertion. Further in vitro experiments indicated that the c.443-3C>G variant in FBN1 was pathogenic and functionally harmful. CONCLUSION: This research identified a novel intronic pathogenic FBN1: c.443-3C>G gene variant, which led to two different aberrant splicing effects. Further functional analysis expands the variant spectrum and provides a strong indication and sufficient basis for preimplantation genetic testing for monogenic disease (PGT-M).

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HLA-B*49:01:01:22 differs from HLA-B*49:01:01:01 by a single nucleotide C->G change in intron 5 at gDNA 2451.

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The process of splicing messenger RNA to remove introns plays a central role in creating genes and gene variants. We describe Splam, a novel method for predicting splice junctions in DNA using deep residual convolutional neural networks. Unlike previous models, Splam looks at a 400-base-pair window flanking each splice site, reflecting the biological splicing process that relies primarily on signals within this window. Splam also trains on donor and acceptor pairs together, mirroring how the splicing machinery recognizes both ends of each intron. Compared to SpliceAI, Splam is consistently more accurate, achieving 96% accuracy in predicting human splice junctions.

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Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC1-244) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes.

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Alternative splicing (AS) in human genes is widely viewed as a mechanism for enhancing proteomic diversity. AS can also impact gene expression levels without increasing protein diversity by producing 'unproductive' transcripts that are targeted for rapid degradation by nonsense-mediated decay (NMD). However, the relative importance of this regulatory mechanism remains underexplored. To better understand the impact of AS-NMD relative to other regulatory mechanisms, we analyzed population-scale genomic data across eight molecular assays, covering various stages from transcription to cytoplasmic decay. We report threefold more unproductive splicing compared with prior estimates using steady-state RNA. This unproductive splicing compounds across multi-intronic genes, resulting in 15% of transcript molecules from protein-coding genes being unproductive. Leveraging genetic variation across cell lines, we find that GWAS trait-associated loci explained by AS are as often associated with NMD-induced expression level differences as with differences in protein isoform usage. Our findings suggest that much of the impact of AS is mediated by NMD-induced changes in gene expression rather than diversification of the proteome.

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Pyrethroid are the primary insecticides used for controlling of Bactricera dorsalis, a highly destructive and invasive fruit pest. Field populations have developed serious resistance, especially to ß-cypermethrin. While mutations in the voltage-gated sodium channel (Vgsc) are a common mechanism of pyrethroid resistance, variations in BdVgsc associated with ß-cypermethrin resistance remain unclear. Here, we reported the resistance levels of five field populations from China, with resistance ratio ranging from 1.54 to 21.34-fold. Cloning the full length of BdVgsc revealed no specific or known amino acid mutations between the most resistant population and the susceptible strain. However, three types of partial intron retention (IRE4-5, IRE19-f and IREL-24) were identified in BdVgsc transcripts, with these intron retentions containing stop codons. The expression of IRE4-5 transcripts and total BdVgsc showed different trends across developmental stages and tissues. Exposure to ß-cypermethrin led to increased expression of IRE4-5. Comparison of genomic and transcriptional sequences reveled that IRE4-5 transcripts had two types (IRE4-5.5 T and IRE4-5.6 T) caused by genomic variations. Both field and congenic strains indicated that homozygotes for IRE4-5.5 T had lower IRE4-5 transcript levels than homozygotes for IRE4-5.6 T. However, congenic and field strains exhibited inconsistent results about the association of expression levels of IRE4-5 transcripts with sensitivity to ß-cypermethrin. In summary, this study is the first to identify intron retention transcripts in the Vgsc gene from B. dorsalis and to examine their expression patterns across different developmental stages, tissues, and strains with varying sensitivities to ß-cypermethrin. The potential role of the intron retentions of BdVgsc in insecticide toxicity is also discussed.

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In vitro circular RNA (circRNA) preparation methods have been gaining a lot of attention recently as several reports suggest that circRNAs are more stable, with better performances in cells and in vivo, than linear RNAs in various biomedical applications. Self-splicing ribozymes are considered a major in vitro circRNA generation method for biomedical applications due to their simplicity and efficiency in the circularization of the gene of interest. This review summarizes, updates, and discusses the recently developed self-circularization methods based on the self-splicing ribozyme, such as group I and II intron ribozymes, and the pros and cons of each method in preparing circRNA in vitro.

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Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting fatty acid and amino acid oxidation with an incidence of 1 in 200,000 live births. MADD has three clinical phenotypes: severe neonatal-onset with or without congenital anomalies, and a milder late-onset form. Clinical diagnosis is supported by urinary organic acid and blood acylcarnitine analysis using tandem mass spectrometry in newborn screening programs. MADD is an autosomal recessive trait caused by biallelic mutations in the ETFA, ETFB, and ETFDH genes encoding the alpha and beta subunits of the electron transfer flavoprotein (ETF) and ETF-coenzyme Q oxidoreductase enzymes. Despite significant advancements in sequencing techniques, many patients remain undiagnosed, impacting their access to clinical care and genetic counseling. In this report, we achieved a definitive molecular diagnosis in a newborn by combining whole-genome sequencing (WGS) with RNA sequencing (RNA-seq). Whole-exome sequencing and next-generation gene panels fail to detect variants, possibly affecting splicing, in deep intronic regions. Here, we report a unique deep intronic mutation in intron 1 of the ETFDH gene, c.35-959A>G, in a patient with early-onset lethal MADD, resulting in pseudo-exon inclusion. The identified variant is the third mutation reported in this region, highlighting ETFDH intron 1 vulnerability. It cannot be excluded that these intronic sequence features may be more common in other genes than is currently believed. This study highlights the importance of incorporating RNA analysis into genome-wide testing to reveal the functional consequences of intronic mutations.

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Beauveria bassiana has potential for Aedes aegypti biological control. However, its efficacy depends on the strain's geographic location, host susceptibility, and virulence. The present study aimed to evaluate the effectiveness of B. bassiana strain BBPTG4 conidia in controlling Ae. aegypti adults and its detection via introns profile on exposed mosquito corpses. Morphologic characteristics among strains were highly similar. Comprehensive testing of these strains demonstrated that BBPT4 exhibited the ideal biological activity for Ae. aegypti control, with a median lethal time (TL50) of 7.5 d compared to ~3 d and ~10 d for BB01 and BB37 strains, respectively. Infected mosquitoes died after GHA and BBPTG4 exposure, and corpses were analyzed for infecting strains detection. Differences among the seven evaluated strains were determined, assessing five different insertion group I intron profiles in BBTG4, BB01, GHA, BB37, and BB02 strains. Mosquitoes infected by BBPTG4 and non-exposed (negative control) intron profiles were obtained. We detected the presence of introns in the BBPTG4 strain, which were not present in non-exposed mosquitoes. In conclusion, B. bassiana strains showed similarities in terms of their cultural and microscopic morphological characteristics and biologicals virulence level, but different intron profiles. BBPTG4 strain-infected Ae. aegypti adult corpses, showing specific amplicons, enabled us to identify B. bassiana at the strain level among infected mosquitoes. However, monitoring and detection of field-infected insects is essential for further verification.

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Limited exploration in fungal mitochondrial genetics has uncovered diverse inheritance modes. The mitochondrial genomes are inherited uniparentally in the majority of sexual eukaryotes, our discovery of persistent mitochondrial heterogeneity within the natural population of the basidiomycete fungus Thelephora ganbajun represents a significant advance in understanding mitochondrial inheritance and evolution in eukaryotes. Here, we present a comprehensive analysis by sequencing and assembling the complete mitogenomes of 40 samples exhibiting diverse cox1 heterogeneity patterns from various geographical origins. Additionally, we identified heterogeneous variants in the nad5 gene, which, similar to cox1, displayed variability across multiple copies. Notably, our study reveals a distinct prevalence of introns and homing endonucleases in these heterogeneous genes. Furthermore, we detected potential instances of horizontal gene transfer involving homing endonucleases. Population genomic analyses underscore regional variations in mitochondrial genome composition among natural samples exhibiting heterogeneity. Thus, polymorphisms in heterogeneous genes, introns, and homing endonucleases significantly influence mitochondrial structure, structural variation, and evolutionary dynamics in this species. This study contributes valuable insights into mitochondrial genome architecture, population dynamics, and the evolutionary implications of mitochondrial heterogeneity in sexual eukaryotes.

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Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription-polymerase chain reaction (RT-PCR)-Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.

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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.

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While Acanthella acuta Schmidt 1862, a common demosponge found in the Mediterranean Sea and Atlantic Ocean, is morphologically similar to other sponges, its mitochondrial DNA (mtDNA) is unique within the class. In contrast to all other studied demosponges, the mtDNA of A. acuta is inferred to be linear and displays several unusual features such as inverted terminal repeats, group II introns in three mitochondrial genes, and two unique open reading frames (ORFs): one of which (ORF1535) combines a DNA polymerase domain with a DNA-directed RNA polymerase domain, while the second bears no discernible similarity to any reported sequences. The group II intron within the cox2 gene is the first such intron reported in an animal. Our phylogenetic analyses indicate that the cox1 intron is related to similar introns found in other demosponges, while the cox2 intron is likely not of animal origin. The two domains found within ORF1535 do not share a common origin and, along with the cox2 intron, were likely acquired by horizontal gene transfer. The findings of this paper open new avenues of exploration in the understanding of mtDNA linearization within Metazoa.

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The study of species groups in which the presence of interspecific hybridization or introgression phenomena is known or suspected involves analysing shared bi-parentally inherited molecular markers. Current methods are based on different categories of markers among which the classical microsatellites or the more recent genome wide approaches for the analyses of thousands of SNPs or hundreds of microhaplotypes through high throughput sequencing. Our approach utilizes intron-targeted amplicon sequencing to characterise multi-locus intron polymorphisms (MIPs) and assess genetic diversity. These highly variable intron regions, combined with inter-specific transferable loci, serve as powerful multiple-SNP markers potentially suitable for various applications, from species and hybrid identification to population comparisons, without prior species knowledge. We developed the first panel of MIPs highly transferable across fish genomes, effectively distinguishing between species, even those closely related, and populations with different structures. MIPs offer versatile, hypervariable nuclear markers and promise to be especially useful when multiple nuclear loci must be genotyped across different species, such as for the monitoring of interspecific hybridization. Moreover, the relatively long sequences obtained ease the development of single-locus PCR-based diagnostic markers. This method, here demonstrated in teleost fishes, can be readily applied to other taxa, unlocking a new source of genetic variation.

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Severe congenital neutropenia (SCN) comprises a diverse range of rare hematological disorders characterized by recurrent, often life-threatening infections that manifest within the first months of life. Mutations in the ELANE gene are the most prevalent cause of SCN. While over 230 mutations in ELANE have been documented, including substitutions, frameshifts, nonsense mutations, and splice site alterations, the occurrence of deep intronic mutations has not been previously reported. Herein, we present the case of a young girl who exhibited recurrent fever, respiratory infections, skin abscesses, and gingivitis shortly after birth. Laboratory analysis revealed markedly diminished neutrophil levels alongside elevated monocyte and eosinophil counts. Bone marrow examination disclosed a halt in myelopoiesis maturation. ELANE gene full-length sequencing identified a novel de novo deep intron mutation in ELANE (c.598 + 79G > T), subsequently confirmed by Sanger sequencing. cDNA sequencing of the patient demonstrated aberrant gene splicing. Utilizing a mini-gene splicing assay for ELANE intronic variants, we identified a mutant ELANE allele (c.597 + 1_597 + 83ins) leading to the creation of a premature termination codon (p.Gly200ValfsTer40). Confocal microscopy revealed heightened expression of myeloperoxidase and neutrophil elastase in the patient, suggesting a potential role for the unfolded protein response in the pathogenesis of the deep intron ELANE mutation. In summary, our findings illustrate the first reported instance of de novo deep intron ELANE mutations associated with SCN, underscoring the importance of exploring deep intronic regions in SCN patients lacking identifiable disease-causing gene mutations.

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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.

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Dystrophinopathies are a group of neuromuscular disorders, inherited in an X-linked recessive manner, caused by pathogenic variants in the DMD gene. Copy number variation detection and next generation sequencing allow the detection of around 99 % of the pathogenic variants. However, some patients require mRNA studies from muscle biopsies to identify deep intronic pathogenic variants. Here, we report a child suspected of having Duchenne muscular dystrophy, with a muscle biopsy showing dystrophin deficiency, and negative molecular testing for deletions, duplications, and small variants. mRNA analysis from muscle biopsy revealed a pseudoexon activation that introduce a premature stop codon into the reading frame. gDNA sequencing allowed to identified a novel variant, c.832-186 T>G, which creates a cryptic donor splice site, recognizing the underlying mechanism causing the pseudoexon insertion. This case highlights the usefulness of the mRNA analysis from muscle biopsy when routine genetic testing is negative and clinical suspicion of dystrophinopathies remains the main clinical diagnosis suspicion.

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