Fusion Transcripts Research Articles

Recent advances in the investigation of fusion RNAs and their role in molecular pathology of cancer

Gene fusions have had a significant role in the development of various types of cancer, oftentimes involved in oncogenic activities through dysregulation of gene expression or signalling pathways. Some cancer-associated chromosomal translocations can undergo backsplicing, resulting in fusion-circular RNAs, a more stable isoform immune to RNase degradation. This stability makes fusion circular RNAs a promising diagnostic biomarker for cancer. While the detection of linear fusion RNAs and their function in certain cancers have been described in literature, fusion circular RNAs lag behind due to their low abundance in cancer cells. This review highlights current literature on the role of linear and circular fusion transcripts in cancer, tools currently available for detecting of these chimeric RNAs and their function and how they play a role in tumorigenesis.

Imaging characterization of paediatric tumours with the neurotrophic tyrosine receptor kinase fusion transcript.

The neurotrophic tyrosine receptor kinase (NTRK) fusion transcript (FT) is a major genetic landmark of infantile fibrosarcoma (IFS) and cellular congenital mesoblastic nephroma (cCMN) but is also described in other tumours. The recent availability of NTRK-targeted drugs enhances the need for better identification. We aimed to describe the anatomic locations and imaging features of tumours with NTRK-FT in children. Imaging characteristics of NTRK-FT tumours of 41 children (median age: 4 months; 63% <1 year old; range: 0-188) managed between 2001 and 2019 were retrospectively analysed. The tumours were located in the soft tissues (n = 24, including 19 IFS), kidneys (n = 9, including 8 cCMN), central nervous system (CNS) (n = 5), lung (n = 2), and bone (n = 1). The tumours were frequently deep-located (93%) and heterogeneous (71%) with necrotic (53%) or haemorrhagic components (29%). Although inconstant, enlarged intratumoural vessels were a recurrent finding (70%) with an irregular distribution (63%) in the most frequent anatomical locations. Paediatric NTRK-FT tumours mainly occur in infants with very variable histotypes and locations. Rich and irregular intra-tumoural vascularization are recurrent findings. Apart from IFS of soft tissues and cCMN of the kidneys, others NTRK-FT tumours locations have to be known, as CNS tumours. Better knowledge of the imaging characteristics may help guide the pathological and biological identification.

Cytogenetics in the management of hematological malignancies: An overview of alternative technologies for cytogenetic characterization

Genomic characterization is an essential part of the clinical management of hematological malignancies for diagnostic, prognostic and therapeutic purposes. Although CBA and FISH are still the gold standard in hematology for the detection of CNA and SV, some alternative technologies are intended to complement their deficiencies or even replace them in the more or less near future. In this article, we provide a technological overview of these alternatives. CMA is the historical and well established technique for the high-resolution detection of CNA. For SV detection, there are emerging techniques based on the study of chromatin conformation and more established ones such as RTMLPA for the detection of fusion transcripts and RNA-seq to reveal the molecular consequences of SV. Comprehensive techniques that detect both CNA and SV are the most interesting because they provide all the information in a single examination. Among these, OGM is a promising emerging higher-solution technique that offers a complete solution at a contained cost, at the expense of a relatively low throughput per machine. WGS remains the most adaptable solution, with long-read approaches enabling very high-resolution detection of CAs, but requiring a heavy bioinformatics installation and at a still high cost. However, the development of high-resolution genome-wide detection techniques for CAs allows for a much better description of chromoanagenesis. Therefore, we have included in this review an update on the various existing mechanisms and their consequences and implications, especially prognostic, in hematological malignancies.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPSeq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPSeq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells.

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPSeq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPSeq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

Droplet digital PCR for sensitive relapse detection in acute myeloid leukaemia patients transplanted by reduced intensity conditioning.

Follow-up after allogeneic transplantation in acute myeloid leukaemia (AML) is guided by measurable residual disease (MRD) testing. Quantitative polymerase chain reaction (qPCR) is the preferred MRD platform but unfortunately, 40%-60% of AML patients have no high-quality qPCR target. This study aimed to improve MRD testing by utilising droplet digital PCR (ddPCR). ddPCR offers patient-specific monitoring but concerns of tracking clonal haematopoiesis rather than malignant cells prompt further validation. Retrospectively, we performed MRD testing on blood and bone marrow samples from AML patients transplanted by reduced-intensity conditioning. The applicability of ddPCR was 39/42 (92.9%). Forty-five ddPCR assays were validated with a 0.0089% median sensitivity. qPCR targeting NPM1 mutation detected relapse 46 days before ddPCR (p = .03). ddPCR detected relapse 34.5 days before qPCR targeting WT1 overexpression (p = .03). In non-relapsing patients, zero false positive ddPCR MRD relapses were observed even when monitoring targets associated with clonal haematopoiesis such as DNMT3A, TET2, and ASXL1 mutations. These results confirm that qPCR targeting NPM1 mutations or fusion transcripts are superior in MRD testing. In the absence of such targets, ddPCR is a promising alternative demonstrating (a) high applicability, (b) high sensitivity, and (c) zero false positive MRD relapses in non-relapsing patients.

IGH::NSD2 Fusion Gene Transcript as Measurable Residual Disease Marker in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy. Approximately 15% of MM patients are affected by the t(4;14) translocation resulting in the IGH::NSD2 fusion transcript. Breakage occurs in three major breakpoint regions within the NSD2 gene (MB4-1, MB4-2, and MB4-3), where MB4-1 leads to the production of full-length protein, while truncated proteins are expressed in the other two cases. Measurable residual disease (MRD) has been conclusively established as a crucial prognostic factor in MM. The IGH::NSD2 fusion transcript can serve as a sensitive MRD marker. Using bone marrow (BM) and peripheral blood (PB) samples from 111 patients, we developed a highly sensitive quantitative real-time PCR (qPCR) and digital PCR (dPCR) system capable of detecting fusion mRNAs with a sensitivity of up to 1:100,000. PB samples exhibited sensitivity three orders of magnitude lower compared to BM samples. Patients with an MB4-2 breakpoint demonstrated significantly reduced overall survival (p = 0.003). Our novel method offers a simple and sensitive means for detecting MRD in a substantial proportion of MM patients. Monitoring may be carried out even from PB samples. The literature lacks consensus regarding survival outcomes among patients with different NSD2 breakpoints. Our data align with previous findings indicating that patients with the MB4-2 breakpoint type tend to exhibit unfavorable overall survival.

Design, Synthesis, and Evaluation of New 1H-Benzo[d]imidazole Based PqsR Inhibitors as Adjuvant Therapy for Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor 1 (IC50 3.2 μM in P. aeruginosa PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled PpqsA-lux transcriptional reporter fusion in P. aeruginosa at low submicromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)-quinolones production.

Dual-Fluorescence Chromosome-Located Labeling System for Accurate In Vivo Single-Cell Gene Expression Analysis in Pseudomonas syringae.

Epigenetic regulation as a means for bacterial adaptation is receiving increasing interest in the last decade. Significant efforts have been directed towards understanding the mechanisms giving raise to phenotypic heterogeneity within bacterial populations and its adaptive relevance. Phenotypic heterogeneity mostly refers to phenotypic variation not linked to genetic differences nor to environmental stimuli. Recent findings on the relevance of phenotypic heterogeneity on some bacterial complex traits are causing a shift from traditional assays where bacterial phenotypes are defined by averaging population-level data, to single-cell analysis that focus on bacterial individual behavior within the population. Fluorescent labeling is a key asset for single-cell gene expression analysis using flow cytometry, fluorescence microscopy, and/or microfluidics.We previously described the generation of chromosome-located transcriptional gene fusions to fluorescent reporter genes using the model bacterial plant pathogen Pseudomonas syringae. These fusions allow researchers to follow variation in expression of the gene(s) of interest, without affecting gene function. In this report, we improve the analytic power of the method by combining such transcriptional fusions with constitutively expressed compatible fluorescent reporter genes integrated in a second, neutral locus of the bacterial chromosome. Constitutively expressed fluorescent reporters allow for the detection of all bacteria comprising a heterogeneous population, regardless of the level of expression of the concurrently monitored gene of interest, thus avoiding the traditional use of stains often incompatible with samples from complex contexts such as the leaf.

A Protocol for the Detection of Fusion Transcripts Using RNA-Sequencing Data.

Fusion transcripts are formed when two genes or their mRNAs fuse to produce a novel gene or chimeric transcript. Fusion genes are well-known cancer biomarkers used for cancer diagnosis and as therapeutic targets. Gene fusions are also found in normal physiology and lead to the evolution of novel genes that contribute to better survival and adaptation for an organism. Various in vitro approaches, such as FISH, PCR, RT-PCR, and chromosome banding techniques, have been used to detect gene fusion. However, all these approaches have low resolution and throughput. Due to the development of high-throughput next-generation sequencing technologies, the detection of fusion transcript becomes feasible using whole genome sequencing, RNA-Seq data, and bioinformatics tools. This chapter will overview the general computational protocol for fusion transcript detection from RNA-sequencing datasets.

A whole-cell hypersensitive biosensor for beta-lactams based on the AmpR-AmpC regulatory circuit from the Antarctic Pseudomonas sp. IB20.

Detecting antibiotic residues is vital to minimize their impact. Yet, existing methods are complex and costly. Biosensors offer an alternative. While many biosensors detect various antibiotics, specific ones for beta-lactams are lacking. To address this gap, a biosensor based on the AmpC beta-lactamase regulation system (ampR-ampC) from Pseudomonas sp. IB20, an Antarctic isolate, was developed in this study. The AmpR-AmpC system is well-conserved in the genus Pseudomonas and has been extensively studied for its involvement in peptidoglycan recycling and beta-lactam resistance. To create the biosensor, the ampC coding sequence was replaced with the mCherry fluorescent protein as a reporter, resulting in a transcriptional fusion. This construct was then inserted into Escherichia coli SN0301, a beta-lactam hypersensitive strain, generating a whole-cell biosensor. The biosensor demonstrated dose-dependent detection of penicillins, cephalosporins and carbapenems. However, the most interesting aspect of this work is the high sensitivity presented by the biosensor in the detection of carbapenems, as it was able to detect 8 pg/mL of meropenem and 40 pg/mL of imipenem and reach levels of 1-10 ng/mL for penicillins and cephalosporins. This makes the biosensor a powerful tool for the detection of beta-lactam antibiotics, specifically carbapenems, in different matrices.

Genetic Approaches to Study the Interplay Between Transcription and Nucleoid-Associated Proteins in Escherichia coli.

Bacterial nucleoid-associated proteins are important factors in regulation of transcription, in nucleoid structuring, and in homeostasis of DNA supercoiling. Vice versa, transcription influences DNA supercoiling and can affect DNA binding of nucleoid-associated proteins (NAPs) such as H-NS in Escherichia coli. Here we describe genetic tools to study the interplay between transcription and nucleoid-associated proteins in E. coli. These methods include construction of genomic and plasmidic transcriptional and translational lacZ reporter gene fusions to study regulation of promoters; insertion of promoter cassettes to drive transcription into a locus of interest in the genome, for example, an H-NS-bound locus; and construction of isogenic hns and stpA mutants and precautions in doing so.

Investigation of sRNA-mRNA Interactions in Bacillus subtilis In Vivo.

In this chapter, we describe in vivo methods for the analysis of interactions between an sRNA and its target mRNA in B. subtilis. All these methods have been either established or significantly improved in our group and successfully employed to characterize a number of sRNA/target mRNA systems in Bacillus subtilis. Whereas in Chap. 8, we describe a combination of in vitro methods, e.g., EMSA and RNA secondary structure probing, we focus here on the investigation of RNA-RNA interactions in vivo using compatible plasmids or chromosomal insertions and deletions, the elucidation of the mechanisms of action of regulatory sRNAs employing transcriptional and translational reporter gene fusions, as well as the determination of expression profiles, half-lives of sRNA and mRNA, and their intracellular concentrations, and, finally, the investigation of RNA chaperones that promote the sRNA/mRNA interaction. For an in-depth analysis of sRNA-mRNA interactions in B. subtilis, a combination of in vivo and in vitro methods should be applied.

ZNF384 fusion transcript levels for measurable residual disease monitoring in adult B-cell acute lymphoblastic leukemia.

Zinc finger protein 384 (ZNF384) rearrangement defined a novel subtype of B-cell acute lymphoblastic leukemia (B-ALL). The prognostic significance of ZNF384 fusion transcript levels represented measurable residual disease remains to be explored. ZNF384 fusions were screened out in 57 adult B-ALL patients at diagnosis by real-time quantitative polymerase chain reaction and their transcript levels were serially monitored during treatment. The reduction of ZNF384 fusion transcript levels at the time of achieving complete remission had no significant impact on survival, whereas its ≥2.5-log reduction were significantly associated with higher relapse free survival (RFS) and overall survival (OS) rates after course 1 consolidation (p=0.022 and=0.0083) and course 2 consolidation (p=0.0025 and=0.0008). Compared with chemotherapy alone, allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly improved RFS and OS of patients with <2.5-log reduction after course 1 consolidation (p<0.0001 and=0.0002) and course 2 consolidation (p=0.0003 and=0.019), whereas exerted no significant effects in patients with ≥2.5-log reduction (all p>0.05). ZNF384 fusion transcript levels after course 1 and course 2 consolidation strongly predict relapse and survival and may guide whether receiving allo-HSCT in adult B-ALL.

MBNL2-ALK: A novel ALK fusion transcript in a sinonasal melanoma

MBNL2-ALK: A novel ALK fusion transcript in a sinonasal melanoma

From intestine to beyond: Salmonella entry factors display distinct transcription pattern upon infection in murine models.

The infectious process of bacteria of the genus Salmonella requires the finely regulated use of various virulence factors. Among them, the type 3 secretion system-1 (T3SS-1) and the Rck and PagN invasins are involved in the internalization of the pathogen within eukaryotic cells, but their precise role in the host and in the pathogenic process is still poorly understood. In this study, we aimed to determine the kinetics of expression of these entry factors in a typhoid fever-like and a gastroenteritis model in mice by in vivo imaging using bioluminescent Salmonella Typhimurium reporter strains carrying chromosomal transcriptional fusions. Only pagN and T3SS-1 transcription has been clearly identified. Independently of the pathological model, the caecum was identified as the main transcription site of both pagN and the T3SS-1-encoding gene both at early and late stages of the infection. An intense transcription of pagN was also observed in deep organs in the typhoid fever-like model, while that of T3SS-1 remained quite sporadic in these organs, and mainly focused on the intestine all along the infection. This work will help to understand the respective role of these entry factors at the cellular level in the pathogenesis of Salmonella in vivo.

Improved detection of clinically relevant fusion transcripts in cancer by machine learning classification

BackgroundGenomic rearrangements in cancer cells can create fusion genes that encode chimeric proteins or alter the expression of coding and non-coding RNAs. In some cancer types, fusions involving specific kinases are used as targets for therapy. Fusion genes can be detected by whole genome sequencing (WGS) and targeted fusion panels, but RNA sequencing (RNA-Seq) has the advantageous capability of broadly detecting expressed fusion transcripts.ResultsWe developed a pipeline for validation of fusion transcripts identified in RNA-Seq data using matched WGS data from The Cancer Genome Atlas (TCGA) and applied it to 910 tumors from 11 different cancer types. This resulted in 4237 validated gene fusions, 3049 of them with at least one identified genomic breakpoint. Utilizing validated fusions as true positive events, we trained a machine learning classifier to predict true and false positive fusion transcripts from RNA-Seq data. The final precision and recall metrics of the classifier were 0.74 and 0.71, respectively, in an independent dataset of 249 breast tumors. Application of this classifier to all samples with RNA-Seq data from these cancer types vastly extended the number of likely true positive fusion transcripts and identified many potentially targetable kinase fusions. Further analysis of the validated gene fusions suggested that many are created by intrachromosomal amplification events with microhomology-mediated non-homologous end-joining.ConclusionsA classifier trained on validated fusion events increased the accuracy of fusion transcript identification in samples without WGS data. This allowed the analysis to be extended to all samples with RNA-Seq data, facilitating studies of tumor biology and increasing the number of detected kinase fusions. Machine learning could thus be used in identification of clinically relevant fusion events for targeted therapy. The large dataset of validated gene fusions generated here presents a useful resource for development and evaluation of fusion transcript detection algorithms.

Panel-based RNA fusion sequencing improves diagnostics of pediatric acute myeloid leukemia

New methods like panel-based RNA fusion sequencing (RNA-FS) promise improved diagnostics in various malignancies. We here analyzed the impact of RNA-FS on the initial diagnostics of 241 cases with pediatric acute myeloid leukemia (AML). We show that, compared to classical cytogenetics (CCG), RNA-FS reliably detected risk-relevant fusion genes in pediatric AML. In addition, RNA-FS strongly improved the detection of cryptic fusion genes like NUP98::NSD1, KMT2A::MLLT10 and CBFA2T3::GLIS2 and thereby resulted in an improved risk stratification in 25 patients (10.4%). Validation of additionally detected non-risk-relevant high confidence fusion calls identified PIM3::BRD1, C22orf34::BRD1, PSPC1::ZMYM2 and ARHGAP26::NR3C1 as common genetic variants and MYB::GATA1 as recurrent aberration, which we here describe in AML subtypes M0 and M7 for the first time. However, it failed to detect rare cytogenetically confirmed fusion events like MNX1::ETV6 and other chromosome 12p-abnormalities. As add-on benefit, the proportion of patients for whom measurable residual disease (MRD) monitoring became possible was increased by RNA-FS from 44.4 to 75.5% as the information on the fusion transcripts’ sequence allowed the design of new MRD assays.

Strategies for validation of a low-volume BCR::ABL p190 assay in the clinical laboratory

Abstract Background The low prevalence of samples positive for the BCR::ABL p190 (e1a2) fusion transcript (&amp;lt;1/week) can be a barrier for molecular test validation. The Cepheid Xpert BCR-ABL Ultra p190 Assay is a single-use cartridge-based RUO assay designed for the quantitative detection of BCR::ABL p190 from fresh specimens. The following study describes our validation strategy to overcome this barrier for implementing a quantitative molecular LDT. Methods This study was conducted in a hospital clinical laboratory setting between 2019 to 2023. RNA was extracted and frozen from peripheral blood (PB) and bone marrow (BM) samples that were sent in parallel to a reference lab for BCR::ABL p190 testing. High positive diagnostic specimens (&amp;gt;10% p190/ABL1) were presumptively identified as BCR::ABL FISH positive and p210 negative. Diagnostic specimens were diluted in known negative PB or BM to achieve %p190/ABL1 in various quantitative ranges. Reverse transcription, amplification, and quantitative real-time PCR were performed in a single reaction format (Cepheid Xpert cartridge®). For accuracy determinations, reported results of clinical samples from the reference lab were compared to the obtained values from our validation using extracted RNA only. Banked proficiency testing (PT) material was used to supplement qualitative and quantitative accuracy. For precision, Invivoscribe (IVS) control material (San Diego, CA) diluted in seven levels (12.5% to 0.0031%; 4-fold increments) and Maine Molecular Quality Controls (MMQCI, Saco ME) at 10%, 1%, 0.1%, 0.02%, 0% were run by at least three technologists and rotated amongst the 16 instrument modules. To evaluate linearity, IVS controls at 15 levels (100% to 0.003%; 2-fold increments) were assayed in triplicate. Limit of detection (LoD) was the minimum concentration at which the detection rate is 100% in 10 independent runs. For analytical specificity, high-positive p210 specimens (&amp;gt;10%) were evaluated for detection of p190. Results a total of 169 samples were analyzed (33 BM, 32 PB, 5 PT samples, 73 IVS controls, and 26 MMQCI controls). We found a correlation (linearity) between reference lab results and clinical/ control samples (R-squared ~0.88, P &amp;lt; 0.01). Overall Cohen’s kappa concordance for qualitative accuracy analysis was 0.80 (CI 95% 0.65-0.95). Sensitivity, specificity, and accuracy were 91%, 92%, and 91%, respectively. This assay showed precision in all levels of dilution (IVS and MMQCI samples) as previously established. LoD was 0.02%. Samples with high level of p210 /ABL remained negative with the p190 assay. Conclusion in the setting of low-volume assays optimized for fresh samples, such as for the detection of BCR::ABL p190, dilutions of clinical samples, banked frozen RNA extracts, PT samples, and commercial controls are important tools for validation of a quantitative LDT.

Somatic and Germline Cohesin Genes Alterations in Pediatric Acute Lymphoblastic Leukemia

Leukemia is a complex disease and the molecular mechanisms of malignant transformation are not fully understood yet. Cohesins are essential proteins, forming a complex which plays critical roles in various cellular processes, not only by canonical DNA binding and chromosome segregation but also in non-canonical regulation of gene expression in both proliferating and post-mitotic cells. Mutations or gene expression alterations of genes encoding the components of the cohesins' complex may affect chromosomal stability and potentially contribute to the pathogenesis of leukemia. Somatic cohesins mutations have been already reported to occur in myeloproliferative disorders and pediatric Acute Lymphoblastic Leukemia (ALL), while on the other hand, germline mutations are causative of Cohesinopathies such as Cornelia de Lange Syndrome (CdLS). Our previous first report of a patient affected by both CdLS and ALL suggested a potential involvement of cohesin germline mutations in the pathogenesis of ALL. Moreover, we demonstrated that germline mutations in STAG1 gene could play a crucial role in predisposition to pediatric hematological malignancies, including ALL. These findings suggest that both somatic and germline alterations of cohesin genes may be associated with the development of ALL. This study aims to understand the molecular mechanisms affected by Cohesin genes alterations, that could potentially pave the way for the development of targeted therapies and improved treatment strategies for leukemia patients. By employing a DNA-targeted next-generation sequencing (NGS) screening of a cohort of 120 consecutive pediatric ALL cases (enrolled in the AIEOP-BFM protocol at various Italian centers), we discovered a total of 11 germline cohesin mutations, further categorized as follows: 6 out of 11 were missense, 4 out of 11 were located in the 3' or 5' untranslated regions (UTR) and 1 out of 11 was a splice-acceptor variant. To investigate the functional implications of selected germline mutations in STAG1 gene, we took advantage of the CRISPR/Cas9 technique to generate in vitro models of two Lymphoblastoid Cell Lines (LCLs) harboring specific variants already found in two patients of our cohort. Additionally, we established control LCLs from healthy donors' samples using the same CRISPR/Cas9 approach. Subsequently, we performed RNA sequencing (RNAseq) analysis on these cell lines, and our preliminary findings revealed a total of 619 differentially expressed genes (DEGs) compared to the control group. Many of them are involved in critical intracellular pathways, like ribosomal transcription, suggesting potential disruptions of essential cellular processes caused by germline STAG1 mutations. Moreover, we have successfully developed an advanced computational analysis pipeline with the primary objective of detecting fusion transcripts from RNAseq data. This pipeline was applied to a dataset of 529 consecutive diagnose and 95 relapse samples of pediatric B/T-ALL patients and in five B-ALL cases a fusion transcript involving the STAG1 (n=1), STAG2 (n=3), and NIPBL (n=1) genes was identified. The significance of these findings was further reinforced by additional cases from both pediatric and adult cohorts through an international collaborative effort and five more fusion transcripts of STAG2 (n=2) and NIPBL (n=3) were uncovered. Investigation to clarify the underlying functional consequences of these fusion genes in pediatric ALL are ongoing. Collectively, these observations endeavor to elucidate the role of germline and somatic cohesin events in pediatric ALL. In particular, the investigation seeks to unveil the non-canonical functions of these proteins, thus paving the way for the identification and characterization of a novel and distinct subcategory of leukemia within the pediatric population.