Search Results (295)

Endometriosis is a chronic, estrogen-dependent inflammatory disorder that is increasingly recognized as a systemic condition with profound implications for female reproductive potential. In addition to pelvic distortion and impaired folliculogenesis, growing evidence indicates that intrinsic alterations in oocyte morphology, mitochondrial function, and developmental competence contribute to infertility. The disease is driven by a multifactorial interplay of somatic mutations, epigenetic remodeling, immune dysregulation, and aberrant steroid signaling, which together create a pro-inflammatory, oxidative, and fibrotic microenvironment. Elevated cytokines, reactive oxygen species, and disrupted granulosa-cell function within the follicular niche impair meiotic progression, cytoplasmic maturation, and mitochondrial integrity, potentially accelerating oocyte aging and diminishing reproductive longevity. Epigenetic and post-transcriptional disturbances—including altered DNA methylation, histone modifications, and RNA-splicing defects—further reinforce estrogen dominance, progesterone resistance, and impaired decidualization, with downstream consequences for ovarian–endometrial communication. Although morphological abnormalities have been documented in oocytes from women with endometriosis, clinical outcomes remain heterogeneous, highlighting the need for integrative models that connect molecular alterations to functional reproductive endpoints. A deeper understanding of these mechanisms is essential for identifying biomarkers of oocyte competence and modifiable strategies—ranging from nutritional optimization to reduction of environmental risk factors—in clinical care to safeguard the reproductive potential of women with endometriosis. Full article

RNA dysregulation mediated by aberrant RNA-binding proteins (RBPs) is closely associated with tumorigenesis. However, the tumorigenic mechanisms of each RBP remained unclear. In this study, we demonstrate that downregulation of Splicing factor 3A1 (SF3A1) markedly suppressed the proliferation of colorectal cancer (CRC) cells, with minimal cytotoxicity observed in non-cancerous epithelial cells. The tumor-promoting function of SF3A1 was further validated in an HCT116 xenograft mouse model. Multiple apoptosis assays—including TdT-mediated dUTP nick end labeling (TUNEL) staining, poly-ADP-ribose polymerase (PARP) immunoblotting, and caspase-3/7 activity measurements—showed that SF3A1 inhibited apoptotic signaling in CRC cells. Transcriptome analysis, combined with RNA-immunoprecipitation (RIP), identified Syntaxin 12 (STX12) as a downstream effector of SF3A1. Knockdown of STX12 induced apoptosis in CRC cells but had no effect on the viability of non-cancerous HCEC-1CT epithelial cells. Furthermore, STX12 mRNA levels were significantly reduced following SF3A1 knockdown, indicating that SF3A1-mediated stabilization of STX12 contributes to apoptosis resistance in CRC cells. Collectively, our findings establish that SF3A1 promotes CRC progression by stabilizing STX12 mRNA and selectively inhibiting apoptosis in malignant cells, thereby identifying the SF3A1–STX12 regulatory axis as a novel and selective therapeutic target for CRC. Full article

The neurofibromin 1 (NF1) splice-site mutation c.61-2A>G (rs1131691100) is a rare, pathogenic, autosomal dominant variant that disrupts NF1 tumor-suppressor function, causing neurofibromatosis type 1 (NF1). Its pathogenic mechanism is poorly understood, and the potential for personalized therapeutic genome editing remains unknown due to the absence of a standard framework for investigating splicing disorders. Here, we performed a comprehensive multi-omics analysis of a de novo c.61-2A>G case from South Korea, integrating short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling. We confirm that c.61-2A>G abolishes the canonical splice acceptor site, activating a cryptic splice acceptor 16 nucleotides downstream in exon 2. This splicing shift generates a 16-nucleotide deletion, causing a frameshift and premature stop codon that truncates the protein’s N-terminal region. Long-read sequencing further reveals that the mutation creates a novel CpG dinucleotide, which is methylated in the majority of reads. Finally, we assessed therapeutic correction strategies, revealing that CRISPR-Cas9 prime editing is the only viable approach for in vivo correction. This study provides the first comprehensive multi-omics characterization of the NF1 c.61-2A>G mutation and establishes a minimal framework for precision therapeutic development in silico in monogenic splicing disorders. Full article

Background: Rhabdoid tumor of the kidney (RTK) is a highly aggressive pediatric malignancy characterized by biallelic SMARCB1 loss, resulting in aberrant MYC pathway activation and cell cycle regulation. MYC-activated tumors are vulnerable in splicing functions and sensitive to splicing inhibitors. Therefore, in this study, cyclin-dependent kinase 11 (CDK11), which regulates both cell cycle and RNA splicing, was tested as a therapeutic target in RTK. Methods: CDK11A/B expression was analyzed using the TARGET-RT database. The therapeutic efficacy of the CDK11 inhibitor OTS964 was evaluated in two RTK cell lines (G401 and JMU-RTK-2) and a JMU-RTK-2 xenograft mouse model. Cytotoxicity, apoptosis, cell cycle, and RNA splicing were examined using the Sulforhodamine B assay, immunoblotting, flow cytometry, and RT-PCR. Results: CDK11B, but not CDK11A, was significantly upregulated in RTK and correlated with the poor survival. OTS964 inhibited RTK cell growth in vitro with the IC50 of 33.1 nM (G401) and 19.3 nM (JMU-RTK-2) and significantly prolonged survival in vivo (median survival: 46.5 vs. 37.0 days, p < 0.01) without marked toxicity. Mechanistically, OTS964 induced G2/M cell cycle arrest and p53 upregulation, disrupted RNA splicing via SF3B1 dephosphorylation, and ultimately led to apoptosis through caspase-3 activation. Conclusions: CDK11 inhibition by OTS964 effectively suppresses RTK growth through cell cycle arrest and RNA splicing inhibition, leading to apoptosis. OTS964 shows potent anti-tumor activity and tolerability, supporting CDK11 as a promising therapeutic target for RTK and related SMARCB1-deficient cancers. Full article

Pathogenic germline variants in the ATM gene are associated with a 20–30% lifetime risk of breast cancer. Crucially, a relevant fraction of loss-of-function variants in breast cancer susceptibility genes disrupts pre-mRNA splicing. We aimed to perform splicing analysis of ATM splice-site variants identified in the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing). To this end, we bioinformatically selected 47 splice-site variants across 17 exons that were genetically engineered into three minigenes and assayed in MCF-7 cells. Aberrant splicing was observed in 38 variants. Of these, 30 variants, including 7 missense, yielded no or negligible expression of the minigene full-length (mgFL) transcript. A total of 69 different transcripts were characterized, 48 of which harboured a premature termination codon. Some variants, such as c.2922-1G>A, generated complex patterns with up to 10 different transcripts. Alternative 3′ or 5′ splice-site usage was the predominant event. Integration of ATM minigene read-outs into the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based specifications for the ATM gene enabled the classification of 30 ATM variants as pathogenic or likely pathogenic and 9 as likely benign. Overall, splicing assays provide key information for variant interpretation and the clinical management of patients. Full article

MET exon 14 (METex14) skipping mutations differ from other non-small cell lung cancer (NSCLC) genomic biomarkers as they result in aberrantly spliced MET transcripts and increased MET-signaling. However, the most accurate method for their detection remains debated. We conducted a retrospective study of previously identified METex14 skipping NSCLC samples by using different, commercially available, diagnostic targeted DNA- /RNA-Next-Generation Sequencing (NGS) panels. We primarily used small DNA-NGS panels covering the 5′ splice site of METex14 and supplemented by targeted RNA sequencing for selected cases. Using this approach, we identified <0.2% patients with METex14 mutations. Due to this low frequency, we validated and introduced complementary NGS testing using combined DNA/RNA-panels. This resulted in an increased number of METex14-positive patients (3.5%) and allowed us to identify METex14 skipping transcripts. Collectively, data from our cohort (n = 34) demonstrated that optimal diagnostics of METex14 variants require a complementary DNA-NGS performed with targeted panels covering both METex14 splice sites, and RNA-NGS. Consequently, we propose a new workflow for interpretation of concordant and discordant findings in METex14 detection. Finally, the potential of DNA-identified METex14 variants to cause aberrant splicing was in silico assessed by the MaxEntScan tool, providing a quantitative approach to splicing disruption. Interestingly, we also identified two novel variants located inside METex14, which also produced the METex14 skipping transcript despite being located outside the canonical splice sites. The altered binding site resulting from these exonic mutations was in silico determined by SpliceTransformer. Full article

Congenital Cataracts, Facial Dysmorphism, and Neuropathy (CCFDN) syndrome is a rare autosomal recessive disorder predominantly found among Vlax Roma populations, caused by a deep intronic founder variant in the CTDP1 gene. This review synthesizes recent advances in understanding the molecular mechanisms of CTDP1 dysfunction, highlighting its central role in transcriptional regulation, RNA splicing, DNA repair, and genome integrity. The unique splicing defect caused by the founder disease-causing variant in the Roma population results in a multisystem phenotype with early-onset neuropathy, congenital cataracts, and characteristic facial dysmorphism. Beyond its genetic homogeneity, CCFDN displays variable clinical severity and presents diagnostic challenges due to overlapping syndromic features. We discuss the emerging therapeutic landscape, focusing on antisense oligonucleotides, small molecule modulators, gene replacement, and genome or transcriptome editing strategies, while emphasizing the challenges in targeted delivery and efficacy. Ongoing insights into CTDP1’s broader biological functions and population genetics inform new directions for diagnosis, genetic counselling, and the development of effective therapies for this severe yet underrecognized disorder. Full article

Background/Objectives: Krabbe disease (KD) is a hereditary lysosomal disorder whose hallmark is progressive demyelination, with variable involvement of the central nervous system. It is caused by pathogenic variants in the GALC gene that disrupt the function of its gene product, the lysosomal enzyme galactosylceramidase. We analyzed two unrelated cases (one early infantile and one adult) with a clinical suspicion of KD. Methods: We used a combination of biochemical techniques (high-performance liquid chromatography–tandem mass spectrometry), NGS (resequencing gene panels), splicing assays, and molecular modeling to identify and analyze the pathogenicity of the variants underlying the disorder. Results: The two probands were compound heterozygotes for disease-causing variants in the GALC gene, encoding the lysosomal hydrolase galactosylceramidase. Three of the variants were novel and caused aberrant splicing, either by exon skipping (c.908+5G>A and c.1034-1G>C) or by inclusion of a cryptic, deep intronic pseudoexon (c.621+772G>C). The fourth variant was a known missense change (c.956A>G, p.(Tyr319Cys)) with conflicting interpretations of pathogenicity in the databases. Conclusions: We demonstrated the pathogenicity of the three novel splicing variants, all with strong impact on galactosylceramidase function. We also concluded that the c.956A>G missense variant is a hypomorph usually underlying the later-onset, milder phenotypes of KD. Our results stress the importance of integrated approaches combining clinical, biochemical, and genetic testing to obtain a definitive diagnosis of lysosomal diseases. Full article

Analysis of coding areas has long been used to study monogenic illnesses, but despite the extensive use of whole-exome sequencing (WES), up to half of suspected cases remain genetically unexplained. Variants outside coding areas can alter splicing, transcript stability, or gene regulation, compromising normal gene activity. These include mutations in noncoding RNAs, promoters, enhancers, deep intronic sequences, and untranslated regions (UTRs). Several well-known disorders have been linked to these mechanisms, including β-thalassemia caused by deep intronic mutations leading to aberrant splicing, familial hypercholesterolemia caused by promoter defects affecting LDLR expression, and inherited retinal diseases driven by noncoding variants influencing retinal gene regulation. These instances show that pathogenic variation is not limited to the exome and can have significant clinical implications. This review summarizes current understanding of noncoding and regulatory variants in monogenic diseases, discusses how they influence diagnosis and therapy, and highlights integrative approaches combining genomic, transcriptomic, and epigenomic data. Multi-layered research has increased diagnostic accuracy and unveiled new therapeutic potentials, although noncoding variations make the connection between genotype and phenotype more complex. Noncoding regions will need to be incorporated into standard diagnostic procedures to convert molecular insights into concrete therapeutic applications in the future. Predictive algorithms, patient-derived model systems, and functional validation testing will all help to simplify this process. Full article

Antibody-based therapeutics targeting tumor surface markers have transformed cancer treatment; however, their efficacy is frequently limited by tumor escape mechanisms such as antigen loss, phenotypic switching, and heterogeneous target expression. Beyond genetic or transcriptional changes, RNA alternative splicing (AS) has emerged as a central post-transcriptional mechanism driving antigenic diversity and immune escape. This review outlines how AS-generated isoforms remodel surface antigen structure and function across key therapeutic targets—including CD/19/CD20/CD22, EGFR/HER2, VEGF, and PD-1/PD-L1—thereby promoting resistance to monoclonal antibodies, antibody–drug conjugates, and immune checkpoint inhibitors. The aberrant activity of splicing regulators disrupts canonical exon selection, leading to altered receptor signaling or the secretion of soluble decoy isoforms that evade immune recognition. Emerging therapeutic strategies aim to counteract these processes through antisense oligonucleotide-mediated splicing correction, pharmacologic modulation of splicing regulators, and isoform-selective antibody or CAR-T designs. Collectively, understanding splicing-driven antigenic plasticity reveals an additional, dynamic layer of resistance regulation and provides a framework for developing RNA-informed precision antibody therapies designed to restore antigen expression, overcome immune escape, and enhance durable clinical responses. Full article

Background/Objectives: Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by uncontrolled accumulation of B lymphocytes. A key feature of CLL is the presence of genetic aberrations, particularly alterations of chromosome 17, such as deletion of 17q and/or mutations in the TP53 gene. Since these abnormalities are highly relevant for therapeutic decision-making, assessment of TP53 mutational status is strongly recommended in routine diagnostics. This study aimed to evaluate the reliability of TP53 sequencing results depending on the type of DNA sample analyzed. Methods: DNA was isolated from two different sample types of the same patient: mononuclear cells (CLL1) and purified CD19+ cells (CLL2). The entire coding region of TP53 (exons 2–11), including splice sites (+/− 10 bp), was analyzed using capture-based next-generation sequencing (NGS). Reads were aligned to the GRCh37/hg19 reference genome, and variants were interpreted using DRAGEN Enrichment (Illumina) and Franklin (QIAGEN). Results: In sample CLL1, the NM_000546.6:c.626_627del mutation (Tier I) was identified with a variant allele frequency (VAF) of 57.06%. The same mutation was confirmed in CLL2, but with a higher VAF of 94.78%. Importantly, an additional Tier I mutation (NM_000546.6:c.825_826del) was detected exclusively in CLL2 at a VAF of 1.59%. Both findings met the required sequencing depth as well as coverage per sample, confirming their validity. Conclusions: The study demonstrates that inadequate starting material for DNA isolation may mask low-frequency TP53 mutations, resulting in false-negative results. Accurate detection requires ensuring sufficient CD19+ cell content, which is critical for reliable diagnostics and supports personalized treatment approaches in CLL. Full article

Background: Germline genetic variants impacting splicing are a frequent cause of disease. The clinical interpretation of such variants is challenging for many reasons including the immense complexity of splicing mechanisms. While recent advances in splicing algorithms have improved the accuracy of splice prediction, predicting the nature and abundance of aberrant splicing remains challenging. As RNA testing becomes more mainstream in the clinical diagnostic setting, the complexities of interpretation are coming to light. Methods: Data from patients undergoing concurrent DNA and RNA testing were retrospectively reviewed for unusual splicing impacts to underscore some of these complexities and serve as exemplars in how to avoid pitfalls in the interpretation of sequence variants. Results: Seven rare variants with unusual splicing impacts are presented: a variant at a consensus donor nucleotide position lacking a splice impact (NF1 c.888+2T>C); a mid-exonic missense variant creating a novel donor site and a cryptic acceptor site resulting in pseudo-intronization (BRIP1 c.727A<G p.Ile243Val); one variant creating a spliceosome switch from U12 to U2 (LZTR1 c.2232G>A p.Ala744Ala); two variants that would be expected to result in nonsense-mediated-mRNA-decay triggering splicing impacts that obviated nonsense-mediated-decay (APC c.1042C>T p.Arg348Ter and BRCA2 c.6762del; c.6816_6841+1534del); and two variants causing splicing impacts through pyrimidine tract optimization (NF1 c.5750-184_5750-178dup and ATM c.3480G>T p.Val1160Val). Conclusions: Paired DNA and RNA testing revealed unexpected splice events altering variant interpretation, expanding our knowledge of clinically important splicing mechanisms and highlighting the benefit of RNA testing. Full article

The SLC26A4 gene is one of the key genes involved in the etiology of hearing loss. It encodes pendrin, a transmembrane transporter protein functioning as a multifunctional anion exchanger. About 600 pathogenic SLC26A4 variants are known to cause either nonsyndromic recessive hearing loss (DFNB4) or Pendred syndrome (hearing loss and thyroid dysfunction). While most pathogenic variants occur in coding regions and disrupt pendrin structure or function, about 25% are thought to impair splicing. For many, pathogenicity has been assessed only in silico, with limited experimental confirmation. We identified several novel and rare SLC26A4 variants in patients with hearing loss from the Tyva and Altai Republics (Southern Siberia, Russia). Based on splicing predictions, six variants—intronic c.2034+1G>A, c.1545-168A>G, c.1708-125T>C, c.1708-18T>A, c.1804-31C>T, and exonic c.942A>G—were selected for analysis using a minigene assay. The results of in vitro analysis only partially matched in silico predictions: c.2034+1G>A caused aberrant splicing; c.1708-18T>A led to exon 16 skipping only in a small proportion of transcripts; the remaining variants showed no detectable splicing effect. These findings underscore the need for integrating in silico predictions with in vitro validation to accurately assess the functional impact of genetic variants, enabling their correct interpretation and reliable molecular diagnosis. Full article

RNA maturation, particularly splicing, depends on coordinated actions of RNA-binding proteins through post-transcriptional processing and constitutes a central mechanism of gene regulation. Aberrant splicing is associated with various diseases, including cancer. Here, we show that the CoREST complex, in coordination with c-MYC, transcriptionally regulates a subset of RNA processing genes, including those encoding essential small nuclear ribonucleoproteins (snRNPs) required for proper spliceosome function. Genetic depletion or the pharmacological inhibition of the CoREST complex in melanoma cells disrupted spliceosome activity, leading to widespread changes in alternative mRNA isoform expression and reduced cell viability. These splicing alterations were associated with changes in the 2′-O-methylation (Nm) of U1 snRNA, a modification critical for spliceosomal function. The ectopic expression of the nucleolar protein NOLC1, a downstream target of the CoREST complex and known for its role in ribosomal RNA processing, partially rescued viability, splicing patterns, and U1 snRNA methylation in CoREST-deficient melanoma cells. Conversely, NOLC1 depletion sensitized melanoma cells to the MEK inhibitor trametinib, a clinical drug approved for treating advanced melanoma. Together, these findings uncover a novel CoREST-NOLC1 axis which is a transcriptional regulatory mechanism playing a significant role in RNA splicing, highlighting that NOLC1 is a downstream effector of the CoREST complex and a potential therapeutic target for melanoma treatment. Full article

Treatment paradigms for B-cell lymphomas have evolved significantly in the last decades. Nevertheless, the widespread clinical use of immunotherapy has demonstrated that it invariably leads to the development of resistance. This review outlines the underlying molecular mechanisms of resistance associated with emerging immunotherapeutic strategies, including Chimeric Antigen Receptor (CAR) T cell therapy and bispecific antibodies (BsAbs). In high-grade B-cell lymphomas, nearly 50% of patients progress following CAR T treatment due to host-related factors affecting CAR T cell proliferation and persistence, as well as tumor-intrinsic factors, such as loss of CD19 epitope expression, trogocytosis, and other genomic alterations (e.g., CD19 mutations, chromothripsis, APOBEC mutational activity, and deletions of RHOA). Additional genomic and epigenetic events, including mutations, alternative splicing of CD19, and aberrant promoter methylation, further contribute to resistance. BsAbs, representing an off-the-shelf T-cell-redirecting strategy, have recently shown promising single-agent efficacy with a manageable toxicity profile, predominantly characterized by T cell overactivation syndromes. Similarly to CAR T cell therapy, BsAb resistance arises through diverse mechanisms, such as antigen loss, T cell dysfunction (exhaustion and regulatory T cell activation), tumor-intrinsic alterations (e.g., TP53 mutations and MYC amplifications), and immunosuppressive influences from the tumor microenvironment. These findings underscore the complexity of immune evasion in B-cell lymphomas and highlight the ongoing need to optimize immunotherapeutic strategies and develop combination approaches to overcome resistance. Full article