Accurate ABO blood group typing is essential for transfusion safety. However, ABO subtypes resulting from genetic variation can complicate this process. In contrast to the extensively studied exonic variants in the ABO gene, splice-site variants are rarely reported, and the mechanisms underlying their contribution to ABO subtypes remain poorly characterized. Four unrelated, healthy blood donors exhibiting mixed-field agglutination with anti-A reagents were subjected to Sanger sequencing. Long-range PCR coupled with nanopore sequencing was further performed to resolve the phased haplotype across the entire ABO locus. The impact of the identified variant on splicing was predicted in silico and validated by an in vitro minigene assay; its effects on protein structure were assessed using bioinformatic tools. Short tandem repeat (STR) analysis was also conducted to exclude blood group chimerism. Four unrelated Chinese blood donors exhibiting mixed-field agglutination with anti-A reagents were identified. Sanger sequencing and nanopore sequencing revealed that all four probands harbored a novel c.239 + 6T>C variant in the ABO*A1.01 allele, with short tandem repeat (STR) analysis ruling out chimerism as the cause of the mixed-field agglutination. In silico analysis predicted activation of a cryptic splice site, leading to retention of a 55-bp intronic fragment in the mRNA transcript. Further, the minigene splicing assay validated this predicted aberrant splicing pattern and additionally revealed trace production of the normally spliced transcript. Secondary and three-dimensional structural modeling further predicted that the resulting aberrant transcript encodes a severely truncated and catalytically compromised glycosyltransferase A, providing a molecular basis for the observed A antigen expression defect. In conclusion, a novel ABO variant (c.239 + 6T>C) responsible for the A3 phenotype was identified, and the mechanism underlying this phenotype was elucidated. By elucidating how a splice site variant disrupts glycosyltransferase function and alters antigen expression, this study contributes to a deeper understanding of the immunogenetic basis of ABO variation, with implications for transfusion safety and personalized immunohematology.
Biallelic disease-causing variants in IGHMBP2 cause spinal muscular atrophy with respiratory distress type I (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S). We present 12 unrelated patients with clinically suspected IGHMBP2 -related-disease, each carrying a variant deep in intron 8 of IGHMBP2 (c.1235+1076G>A (n=6), c.1235+450G>A (n=5), and c.1235+894C>A (n=1)), along with a known deleterious variant in trans. To assess aberrant pathogenic splicing induced by these deep intronic variants in a relevant model, patient-derived induced pluripotent stem cells were differentiated into motor neurons (iMNs). Long-read RNA sequencing revealed introduction of different pseudoexons by each variant: c.1235+450G>A (626bp), c.1235+1076G>A (112bp and 77bp) and c.1235+894C>A (182bp). Although each variant utilizes a unique splice acceptor site, they all activate the same cryptic donor site, enabling a therapeutic approach to redirect aberrant splicing for all the variants using a single shared antisense oligonucleotide (ASO). Treatment of iMNs with this single ASO restored full-length IGHMBP2 protein in c.1235+894G>A and c.1235+1076G>A by decreasing the use of the novel acceptor site. In contrast, ASO treatment did not correct the splicing in c.1235+450G>A, suggesting that additional splice correction will be needed for this specific variant. A CRISPR interference screen of IGHMBP2 loss-of-function in iMNs identified ribonucleoprotein complex biogenesis (RNP), and rRNA and tRNA processing as top pathways implicated in motor neuron vulnerability. Proteomics and transcriptomics analysis of successfully treated patient iMNs revealed correction of RNP biogenesis and rRNA processing defects. This study highlights the importance of characterizing deep intronic variants in disease-relevant cells to assist the diagnostic process and inform therapeutics development. Intron 8 of IGHMBP2 is a hotspot for splice activating pathogenic variants causing SMARD1 and CMT2S, which can be targeted with a single antisense oligonucleotide to correct the aberrant splicing, increase protein and restore cellular function in patient derived motor neurons.
Precise intron removal by RNA splicing is essential for faithful gene expression, yet the mechanisms ensuring splicing fidelity in mammals remain unclear. Using a systematic knockdown RNA sequencing (RNA-seq) screen, we uncover widespread splicing errors and identify AQR, SYF1, and SYF3 as cooperative safeguards of 3' splice-site (3'ss) fidelity in human and mouse. These factors act during spliceosome assembly to correct U2AF-mediated misrecognition of non-canonical 3'ss bearing AG dinucleotides embedded within pyrimidine-rich sequences and lacking canonical branch points (BPs), likely through kinetic proofreading. Their loss triggers pervasive 3'ss mis-splicing, resulting in the accumulation of misfolded proteins, proteotoxic stress, unfolded protein response activation, and ultimately cell death and intestinal inflammation. Together, our study reveals a previously unrecognized layer of splicing fidelity control in mammals that links aberrant splice-site selection to proteostasis and inflammation.
This study investigated the effect of single Gαo1 and Gαo2, as well as double Gαo1/2 knockout on the cerebellar anatomy and synapse formation. The alpha subunit of the G protein Go exists in two splice variants. Knockout of certain Gαo subtypes result in strong-mainly motor-deficits in mice, and mutations in the responsible gene locus in humans can result in severe encephalopathies. We aimed to decipher the hitherto incompletely understood contribution of the individual Gαo subunits to the anatomy and synapse formation of the cerebellum. Knockout of Gαo1 reduced the size of the cerebellum by 11%, accompanied by maximal reductions of the molecular layer thickness in the central lobule III (-30%) and molecular layer area in the uvula (-33%). Knockout of Gαo2 increased cerebellar size, molecular layer thickness in central lobule II (+18.6%), and area of the culmen (+37%). Combined deletion of Gαo1 and Gαo2 reduced cerebellar size by 12%, molecular layer thickness and area of the declive (by -27.3% and -23.4%, respectively). Moreover, VGLUT2-positive climbing fiber contacts to Purkinje cells were reduced in Gαo1 knockout mice (on average by 40%). Similarly, VGLUT1 expression was reduced (on average by 17.3%). The knockout of Gαo2 promoted climbing fiber contacts (+14.3% on average, at a maximum of +52.6% in the central lobule II), VGLUT1 was less affected. Double knockout mice exhibited negative effects on VGLUT2 (-35% overall number) and VGLUT1 (-23% on average in expression levels). VGAT-positive synaptic contacts were also diminished for Gαo1 and double knockout (-25% and -31% overall number, respectively) and increased for Gαo2 knockout (+13% on average). In line with this, negative effects on the dendritic outgrowth of Purkinje cells were observed in both Gαo1 -/- and Gαo -/-mice, while knockout of Gαo2 promoted dendrite outgrowth. Taken together, the two Gαo splice variants contrarily contribute to the development of the cerebellum, with Gαo1 representing the dominant subunit.
Formation of the activated human spliceosome (Bact) involves major structural rearrangements, leading to the catalytically active U2/U6 RNA core. This process involves at least two intermediates, pre-Bact-1 and pre-Bact-2, and is regulated by CDK11-mediated phosphorylation of the U2 snRNP protein SF3B1. However, the mechanisms of this essential step are poorly understood. Here we present the cryo-EM structure of a spliceosome stalled - by the CDK11 inhibitor OTS964 - in a previously undescribed early-activated state, termed pre-Bact-OTS, shortly after dissociation of U4 snRNP. In pre-Bact-OTS, the U2-SF3B6 protein is retained in a C-terminal region of the super-helical U2-SF3B1 HEAT domain (SF3B1HEAT) that clamps the U2/branch-site helix. In contrast, in pre-Bact-1, SF3B6 is repositioned to SF3B1's N-terminal HEAT repeats, thereby preventing a steric clash of SF3B6 with PRP8 during the pre-Bact-OTS-to-pre-Bact-1 transition. We infer that the CDK11-mediated phosphorylation of SF3B1 drives the relocation of SF3B6, gating progression to Bact formation. In pre-Bact-OTS, we also located the RNA helicase DHX15 at the N-terminal region of SF3B1HEAT, assisted by the SR140/SPF45/CHERP/SUGP1 protein complex. These results suggest the involvement of DHX15 in kinase-mediated proofreading of the early-activated spliceosome, by competing with CDK11's phosphorylation of SF3B1, and thus with relocation of SF3B6 at SF3B1HEAT.
Prion diseases are fatal neurodegenerative disorders driven by prion protein (PrP) misfolding, and lowering cellular PrP represents a promising therapeutic strategy. Here we report a small-molecule approach that reduces PrP by modulating pre-mRNA splicing of the PRNP gene. Through chemical modification of the clinically approved splicing modulator risdiplam, we generated CP3, the first class of compounds that selectively activate a cryptic exon in PRNP and routes its mRNA product for degradation, reducing PrP by ∼70% in cells. We demonstrate that CP3 activity critically depends on alternative splicing factor Luc7L, revealing a novel requirement for alternative splicing factors in small molecule splicing modulation. Strikingly, co-administration of CP3 with a Luc7L activator PTC258 significantly lowers PrP levels in the brains of transgenic mice. These results establish splicing modulation as a powerful strategy for PrP reduction and highlight the potential of small-molecule cooperativity for therapeutic RNA targeting.
The Epstein-Barr virus (EBV) infects over 95% of adults, establishing lifelong latency and contributing to the development of various malignancies, including Burkitt lymphoma and nasopharyngeal carcinoma. However, the RNA structures regulating the splicing of the critical EBV gene, latent membrane protein 1 (LMP1), remain uncharacterized. To identify these regulatory elements, we applied spliceosome inhibition with RNA probing and sequencing (SIRP-seq) to the BJAB-B1 cell line. By utilizing the spliceosome inhibitor pladienolide B, we enriched pre-mRNA species, enabling the detection of structural features within both the full-length pre-mRNA (LMP1-FL) and an alternatively spliced isoform retaining intron 2 (LMP1-AS). The resulting chemical probing datasets informed the RNA folding algorithms RNAfold and ScanFold to generate the first high-resolution secondary structure models for the LMP1 pre-mRNA, encompassing both exonic and intronic regions. Our results identify 11 novel, thermodynamically stable RNA structures, with several key elements positioned near splice junctions. Notably, three structures (Structures 8, 9, and 10) were identified near the 3' splice site of intron 2, appearing in alternative conformations that may influence splicing accessibility. Furthermore, these structures map to regions containing disease-relevant mutations associated with patient survival in Burkitt lymphoma. This structural framework provides new insights into how LMP1 splicing may be regulated by RNA structure and identifies potential novel therapeutic targets for mitigating EBV-associated diseases.
Pre-mRNA splicing is carried out by the spliceosome, a large and dynamic ribonucleoprotein complex. The spliceosome is known to be stored in nuclear speckles (NS), which are recognized as active subnuclear organelles for splicing. However, it remains poorly understood how spliceosomal protein-protein interactions are functionally coupled to NS organization to maintain splicing robustness in plants. Here, we report the functional significance of a specific interaction between two U4/U6·U5 tri-snRNP components of the spliceosome, STA1 and DOT2, in regulating NS organization, pre-mRNA splicing, and heat stress responses in Arabidopsis. We identified a missense mutation in DOT2 (a Snu66/SART1 homolog) from a genetic suppressor of the PRP6 homolog mutant sta1-1 (named S307). This mutation restored the weakened interaction between STA1 and DOT2 in the sta1-1 mutant background. Genetic, biochemical, and cell biological analyses showed that variation in the strength of the STA1-DOT2 interaction was closely associated with changes in NS formation, splicing efficiency, transcript abundance, as well as growth and heat tolerance. Pharmacological inhibition of STA1-associated NS formation by tubercidin recapitulated sta1-1-like phenotypes and splicing defects, supporting a functional link between NS organization and splicing outcomes. In addition, heat-induced weakening of the STA1-DOT2 interaction was accompanied by reduced NS formation and increased intron retention at the transcriptome-wide level including key heat-responsive transcripts. Based on these observations, we propose that the STA1-DOT2 interaction, likely reflecting the assembly state of the U4/U6·U5 tri-snRNP, may represent one of the heat-sensitive interaction nodes that couple spliceosome assembly to NS organization and splicing robustness under stress conditions.
RNA splicing is a fundamental feature of eukaryotic gene expression that is co-opted by many nuclear-replicating viruses to produce viral transcripts. Recent clinical development of spliceosome-targeting therapeutics has demonstrated that splicing can be safely modulated in vivo, raising the possibility that viral dependence on host RNA processing may represent an exploitable antiviral vulnerability. Here, we show that pharmacologic inhibition of cellular RNA splicing preferentially suppresses the expression of spliced viral transcripts and profoundly impairs replication of multiple splicing-dependent viruses. In human adenovirus, low-dose inhibition of the spliceosome using mechanistically distinct chemical inhibitors, or genetic depletion of SF3B1, disrupted splicing of complex late viral transcripts while largely sparing simple early transcripts. This resulted in impaired viral DNA replication, reduced late protein accumulation, and multi-log decreases in infectious progeny production. Splicing inhibitors similarly impaired RNA splicing and viral replication of DNA virus Herpes Simplex virus 1 and RNA virus influenza A, which both rely on host-dependent RNA processing strategies. In contrast, cytoplasmic RNA viruses lacking spliceosome dependence were unaffected, supporting an on-target mechanism. Together, these findings identify viral RNA processing complexity as a determinant of antiviral sensitivity and establish host spliceosome dependence as a shared and targetable vulnerability across diverse viral families.
Drug resistance, defined as the adaptive tolerance of human diseases and agricultural pests to therapeutic or chemical agents, threatens global health and food security. Alternative splicing (AS), a central posttranscriptional regulatory mechanism, is increasingly recognized as a conserved driver of resistance evolution. However, although AS-mediated resistance has been widely studied in cancer, its role in pesticide and fungicide resistance remains insufficiently explored, limiting cross-disciplinary reuse of mechanistic insights and intervention strategies. This review critically compares the spliceosome-mediated and AS-mediated mechanisms that underlie resistance to anticancer drugs, pesticides and fungicides within a unified framework, with the goal of clarifying how distinct selective pressures shape convergent adaptive resistance across biological kingdoms. We summarize how aberrant AS promotes resistance through target truncation, altered drug metabolism, apoptosis evasion, and rewiring of survival signaling. We further propose a customized Design-Build-Test-Learn (DBTL) roadmap for AS-targeted intervention, integrating long-read sequencing, AI-assisted prediction, splice-modulating therapeutics such as ASOs and CRISPR-based approaches, nanodelivery optimization, and dynamic resistance monitoring. By linking mechanistic comparison with translational implementation, this review provides an interdisciplinary framework for understanding, predicting, and reversing AS-mediated resistance in both clinical and agricultural settings.
Intellectual disability (ID) affects approximately 1%-3% of the population and spans diverse clinical presentations with marked genetic heterogeneity, especially in consanguineous populations where autosomal-recessive ID is common. Despite advances in diagnostic methods, ~50% of individuals with ID remain without a molecular diagnosis. Genome sequencing (GS) can detect variant classes poorly captured by other methodologies, including deep intronic splice changes and structural variants. We performed short-read GS on 38 Iranian autosomal recessive intellectual disability (ARID) families that remained unsolved after exome sequencing (ES) and two phases of reanalysis. Sequencing and variant calling were performed on DRAGEN Bio-IT Platform with GRCh38 and variants were annotated in Golden helix Varseq software and the AnnotSV tool. GS yielded diagnoses in 2 of 38 families (5.3%), identifying a homozygous deep-intronic ATP8A2 variant (c.1473 + 519C > T) that creates a cryptic donor site and a 57-bp pseudoexon, and a homozygous ~103-kb CNTNAP2 deletion removing the in-frame Exon 2. Additionally, GS uncovered a homozygous NCOR1 missense variant that represents a novel candidate gene for ARID, but further studies are required to confirm the gene-disease association. The ATP8A2 and CNTNAP2 variants were uniquely detectable by GS. In conclusion, in a challenging, ES-negative ARID cohort, GS provided an additional ~5.3% diagnostic yield by uncovering a noncoding splice alteration and a large intragenic deletion. These results underscore GS as a valuable, though still modest, tool over ES and highlight significant interpretation challenges in noncoding regions. Continued advances in functional assays and complementary long-read technologies will be essential to further reduce the diagnostic gap in unresolved ID.
Spliceosomal small nuclear RNA (snRNA) U1 and the U2AF heterodimer play critical functions by recognizing the highly conserved GT and AG dinucleotides, respectively, located at the start and at the end of introns. Here, we explore how changing these components contributed to maintaining splicing function in genomes where 95% of introns escape the GT/AG rule. By gaining access to new tunicate genomes, we could reveal that the emergence of non-canonical introns in the Fritillaria borealis lineage coincides with the duplication of U2AF subunits. Our findings indicate that paralogs U2AF1α and U2AF2α have preserved conserved functions, while divergent paralogs U2AF1β and U2AF2β form novel heterodimers that recognize introns with non-canonical 3' ends. The conserved m6A present on U6 snRNA has been considerably reduced in F. borealis, but its U1 snRNA retains a stable 5'-terminal m6A, which is typically suppressed in humans and other chordates. We propose that this unique m6A pattern stabilizes the binding of snRNA to non-canonical 5' splice sites. Although the core components of the spliceosome remain preserved, functional changes implemented through gene duplication and post-transcriptional modifications can significantly broaden the range of target splice sites.
Hereditary cancers arise from germline pathogenic variants that confer increased lifetime cancer risk. In Palestine, the genetic basis of hereditary cancer predisposition remains limited. The objective of this study is to investigate germline variants associated with hereditary cancer susceptibility in Palestinian families with strong cancer history. Families with suspected hereditary breast cancer were recruited, and germline DNA extracted from peripheral blood was first analyzed using a BRCA1/2 panel. Whole-exome sequencing (WES) was subsequently performed in BRCA-negative probands to identify additional candidate hereditary variants. A total of 34 individuals from three unrelated families were included in the study, comprising 8 affected and 26 unaffected individuals. Segregation analysis and in silico functional assessment were conducted to evaluate variant pathogenicity. We identified a splice-site variant in RAD50 (c.2524+3A>G) in Family I, a truncating MSH4 variant (c.328C>T; p.R110X) and a missense STAT6 variant (c.1216C>G; p.L406V) in Family II, and a splice-region PRKAR1A variant (c.973+6T>C) in Family III. These variants are segregated with disease in the respective families and are predicted to affect protein function. Our findings highlight the importance of germline genomic analysis in characterizing hereditary cancer predisposition in underrepresented populations and provide preliminary data for future risk assessment and genetic counseling strategies in Palestine.
The seismic performance of vertical reinforcement discontinuous splice prefabricated shear walls (SGBL prefabricated shear walls) in high-rise buildings is investigated in this study. The failure mechanisms, load-bearing capacity, ductility, and energy dissipation characteristics of these walls are clarified, with particular attention given to the influence of the shear-span ratio and axial compression ratio. A refined model was established using ABAQUS finite element software, and simulations were conducted on individual SGBL wall panels under various shear-span ratios (achieved by adjusting story heights) and axial compression ratios (0.1, 0.3, 0.5). The results demonstrate that the load-bearing capacity of the wall panels increases significantly with the axial compression ratio (Specimens with shear-span ratios of 1.25, 1.5, and 1.75 exhibited increases of 76.8%, 88%, and 82%, respectively). In contrast, ductility and energy dissipation capacity are reduced. When an axial ratio of 0.5 and a shear-span ratio of 1.25 were applied, interlaced diagonal cracks were observed in specimen SW1-0.5. Therefore, the inclusion of inclined reinforcement is recommended in practice to prevent premature brittle failures. Under a constant axial compression ratio, it was found that the load-bearing capacity decreases as the shear-span ratio increases, whereas ductility and energy dissipation capacity are enhanced. Specimen SW3-0.1, characterized by a low axial compression ratio and a large shear-span ratio, exhibited the optimum energy dissipation capacity and ductility. During the simulation, no vertical cracks were observed at the joint interface between the precast panels and the cast-in-situ components, confirming that the structure exhibits excellent integrity and compatibility.
Modulation of splicing is an established therapeutic strategy with clinical applications and potential to target specific exons to influence gene expression. Small-molecule splicing modifiers such as Risdiplam and Branaplam induce inclusion of exons typically skipped due to weak 5' splice sites. Risdiplam preferentially induces exons with an N-3G-2A-1 sequence at the 3' exon end, whereas Branaplam favors A-3G-2A-1-ending exons. However, determinants of specificity remain unclear, as many motif-matching exons are not induced. Here, we investigate the molecular basis of splicing-modulator specificity. Using biochemical assays, transcriptome analyses, and genetic perturbations, we identify sequence-dependent features that determine exon responsiveness to splicing-modulator induction. We further demonstrate that specificity can be reprogrammed through manipulation of U1 snRNA. These findings refine the determinants of splicing-modulator target space and may support identification of additional target exons and compounds.
TRPV4 is a non-selective cation channel of the TRPV family and plays a key role in fibrosis, but its pathological mechanisms in genetically susceptible individuals remain unclear. This study aimed to investigate the potential role of the Trpv4 c.1491+1G>A splice-site mutation in pulmonary fibrosis using a gene-edited mouse model. The mutation was identified in a family with autosomal dominant familial digital arthropathy-brachydactyly (FDAB). A corresponding gene-edited mouse model was generated using CRISPR/Cas9 technology. Histopathological analysis, single-cell RNA sequencing (scRNA-seq), qPCR, Western blot, and immunofluorescence co-staining were employed to assess phenotypic, transcriptomic, and molecular changes in the lungs. The model mice exhibited skeletal abnormalities and multi-organ damage, with pronounced pulmonary fibrosis. In the lung tissues of homozygous mutant (Trpv4-Hom) mice, wild-type Trpv4 expression was significantly reduced, accompanied by thickened alveolar septa, pulmonary congestion, and increased collagen deposition. scRNA-seq revealed a decrease in the proportions of alveolar macrophages and NK cells, while Clara cells, fibroblasts, mesothelial cells, and alveolar type II cells increased. The ALCAM-CD6 and MIF-CD74 signaling axes were significantly upregulated. qPCR confirmed the transcriptional upregulation of Alcam, Cd6, Cd74, and Mif; Western blot further validated the increased protein levels of ALCAM, CD6, and CD74; and immunofluorescence confirmed the enhanced co-localization of MIF and CD74 in lung tissue. The Trpv4 c.1491+1G>A mutation is associated with exacerbated pulmonary fibrosis, altered lung cellular composition, disrupted ALCAM-CD6 immune regulatory pathways and MIF signaling homeostasis, and enhanced pro-fibrotic cell communication. These findings provide novel molecular targets for the development of anti-fibrotic therapies targeting this pathway.
To explore the clinical phenotype and genetic etiology for a Chinese pedigree affected with Hereditary coagulation factor Ⅴ deficiency (FⅤD). A 47-year-old female who visited the First Affiliated Hospital of Wenzhou Medical University in July 2025 and her family members (husband, mother, younger brother and younger brother's wife, elder sister, second elder sister, daughter, son, elder niece, and second niece) were selected as study subjects. Clinical data and family history were retrospectively collected. Peripheral blood samples were collected from the proband and her family members. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor Ⅱ activity (FⅡ:C), coagulation factor Ⅶ activity (FⅦ:C), coagulation factor Ⅷ activity (FⅧ:C), coagulation factor Ⅹ activity (FⅩ:C), and FⅤ activity (FⅤ:C) were measured in all participants using the one-stage clotting assay. FⅤ antigen (FⅤ:Ag) levels in plasma were determined with enzyme-linked immunosorbent assay (ELISA). Genomic DNA was extracted from peripheral blood samples of all participants, and the coding regions of the F5 gene were amplified by PCR and subjected to Sanger sequencing. Candidate variants were verified with multiple online tools to predict their pathogenicity and impact on splicing. Based on guidelines formulated by the American College of Medical Genetics and Genomics (ACMG), the pathogenicity of candidate variants was evaluated. Coagulation function of all participants was evaluated using thrombin generation assay and thromboelastography. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: KY2022-R193). The proband's peripheral blood PT and APTT were 25.6 s and 58.3 s, respectively, while FⅤ:C and FⅤ:Ag were 7% and 6%, respectively, which was in keeping with a phenotype of type I FⅤD. Her brother also showed significantly decreased FⅤ:C and FⅤ:Ag levels. The proband's mother, eldest sister, second sister, daughter, son, eldest niece, second niece had FⅤ:C and FⅤ:Ag approximately 50% of those of normal controls, whereas her husband and sister-in-law showed no obvious abnormality in such indices. Genetic testing revealed that the proband and her brother both harbored compound heterozygous variants of the F5 gene, namely c.6528+3A>T (IVS24+3A>T) and c.6665A>G (p.Asp2222Gly), for which other family members with reduced FⅤ:C and FⅤ:Ag were heterozygous carriers, and those with normal coagulation indices were of the wild-type. Bioinformatics analyses using multiple software indicated that the IVS24+3A>T variant is located at a splice donor site and can significantly weaken the strength of the splice site, potentially leading to exon skipping. Based on the ACMG guidelines, the novel variant is classified as likely pathogenic (PM2_Moderate+PM3_Moderate+PP1_Supporting+PP3_Supporting+PP4_Supporting). Results of thrombin generation assay indicated reduced thrombin generation capacity in the proband, whilst thromboelastography results showed abnormal coagulation characteristics manifested as prolonged coagulation initiation time. Ultimately, both the proband and her younger brother were diagnosed with hereditary type I FⅤD. The IVS24+3A>T and p.Asp2222Gly compound heterozygous variants of the F5 gene probably underlay the pathogenesis of FⅤD in this pedigree. Above finding has enriched the mutational spectrum of the F5 gene and provided a basis for genetic counseling and molecular diagnosis.
BAP1 tumor predisposition syndrome (BAP1-TPDS) is an autosomal dominant syndrome associated with uveal melanoma and multiple systemic malignancies, which is believed to be underreported. Recently, the presence of polydactylous onychopapillomas were deemed an associated phenotype of BAP1-TPDS. Here we describe a case of choroidal melanoma in a patient with polydactylous onychopapilloma, as well as extensive family history of both BAP1-TPDS and relevant nail findings. A retrospective chart review was conducted. A 62-year-old man presented with choroidal melanoma of the left eye. Tumor genetic expression profiling demonstrated Class 2, PRAME-negative status, and next-generation sequencing identified variants in GNAQ and BAP1 (c.123-12_127delTACTTCCCCCAGCCCTG), with the BAP1 splice-site variant present at a high variant allele frequency (91.3%). Physical examination revealed onychopapillomas affecting multiple digits and a family history of malignancy among first-degree relatives. Germline multi-cancer panel testing confirmed a hereditary BAP1 variant, establishing the diagnosis of BAP1-TPDS. The patient underwent plaque brachytherapy with continued tumor regression and is enrolled in multidisciplinary surveillance for BAP1-associated malignancies; genetic testing was recommended for close relatives. This case underscores the importance of combining tumor sequencing with careful phenotypic assessment and family history review. Recognition of polydactylous onychopapillomas may serve as a valuable clinical clue prompting germline testing for BAP1-TPDS in uveal melanoma patients.
Precise regulation of intracellular calcium signaling is essential for neuronal function and depends on tightly coordinated molecular mechanisms. Among them, endoplasmic reticulum-plasma membrane (ER-PM) junctions form dynamic signaling microdomains that support store-operated calcium entry (SOCE), lipid transfer, phosphoinositide signaling, and ion channel regulation. Within this context, stromal interaction molecules STIM1 and STIM2 act as ER calcium sensors with distinct activation properties. STIM1 is recruited during substantial ER calcium depletion and strong neuronal activity, whereas STIM2 responds to subtle calcium fluctuations and supports basal calcium homeostasis. Upon ER calcium depletion, STIM proteins undergo conformational rearrangement, oligomerization, and translocation to ER-PM junctions, where they engage Orai calcium channels to trigger calcium influx and restore ER calcium balance. Although originally characterized in non-excitable cells, the STIM-Orai signaling axis is now recognized as an important regulator of neuronal calcium dynamics. This system exhibits considerable molecular diversity, with multiple STIM and Orai isoforms and splice variants differing in activation thresholds, subcellular localization, and signaling properties. Consequently, neuronal SOCE is not a uniform calcium entry pathway but contributes to specialized functions across dendrites, dendritic spines, and presynaptic compartments. Beyond classical SOCE, STIM proteins also function as multifunctional organizers of ER architecture and ER-PM junctions, linking calcium homeostasis to dendritic spine morphology, receptor trafficking, neurotransmitter release, and synaptic plasticity. Despite significant progress in this field, a comprehensive comparative understanding of the isoform-specific roles of STIM and Orai proteins in neuronal compartments remains limited. This review summarizes current knowledge regarding the functions of STIM1, STIM2, and Orai1-3 in dendrites and synapses, emphasizing their distinct contributions to calcium homeostasis, local signaling microdomains, and activity-dependent versus homeostatic forms of synaptic plasticity. By integrating evidence from genetic, biochemical, and in vivo studies, this review further highlights region-specific and context-dependent effects, unresolved controversies regarding the physiological roles of STIM and Orai, and the consequences of their dysregulation for neuronal function.
With the rise of checkpoint blockade therapies and neoantigen-based vaccines reaching later-stage trials, there is a growing need for computational tools to identify and prioritize neoantigens. pVACtools, initially introduced in 2016, is an open-source informatic suite designed to support basic and translational neoantigen research. pVACtools assists prediction, prioritization, and visualization of neoantigens, as well as design of neoantigen-based therapies. We describe several major advances to pVACtools since the last update: (1) expanded neoantigen quality and safety assessment features, including support for peptide presentation scoring, immunogenicity prediction, anchor residue analysis, reference proteome similarity, percentile score calculation; (2) addition of pVACsplice, a new tool for predicting neoantigens from tumor-specific cis-splicing mutations; (3) addition of pVACbind, a flexible tool that supports noncanonical neoantigen sources; (4) improvement in neoantigen selection strategies; (5) a substantially improved pVACvector algorithm that achieves higher DNA/mRNA vector vaccine design success rates with shorter runtimes; (6) new utilities to support synthetic long peptide vaccine design; (7) extended prediction support for many non-human species; and (8) addition of pVACcompare, a tool to support comparison between two pVACseq results. Together, these updates reinforce pVACtools as the field's most comprehensive toolkit for neoantigen research, from basic discovery to the design and execution of personalized cancer vaccine clinical trials.