Pediatric neuropathology practice in a low- and middle-income country: capacity building through institutional twinning.

Background: Accurate and precise diagnosis is central to treating central nervous system (CNS) tumors, yet tissue diagnosis is often a neglected focus in low- and middle-income countries (LMICs). Since 2016, the WHO classification of CNS tumors has increasingly incorporated molecular biomarkers into the diagnosis of CNS tumors. While this shift to precision diagnostics promises a high degree of diagnostic accuracy and prognostic precision, it has also resulted in increasing divergence in diagnostic and management practices between LMICs and high-income countries (HICs). Pathologists and laboratory professionals in LMICs lack the proper training and tools to join the molecular diagnostic revolution. We describe the impact of a 7-year long twinning program between Canada and Pakistan on pathology services. Methods: During the study period, 141 challenging cases of pediatric CNS tumors initially diagnosed at Aga Khan University Hospital (AKUH), Karachi, were sent to the Hospital for Sick Children in Toronto, Canada (SickKids), for a second opinion. Each case received histologic review and often immunohistochemical staining and relevant molecular testing. A monthly multidisciplinary online tumor board (MDTB) was conducted to discuss the results with pathologists from both institutions in attendance. Results: Diagnostic discordance was seen in 30 cases. Expert review provided subclassification for 53 cases most notably for diffuse gliomas and medulloblastoma. Poorly differentiated tumors benefited the most from second review, mainly because of the resolving power of specialized immunohistochemical stains, NanoString, and targeted gene panel next-generation sequencing. Collaboration with expert neuropathologists led to validation of over half a dozen immunostains at AKUH facilitating diagnosis of CNS tumors. Conclusions: LMIC-HIC Institutional twinning provides much-needed training and mentorship to pathologists and can help in infrastructure development by adopting and validating new immunohistochemical stains. Persistent unresolved cases indicate that molecular techniques are indispensable in for diagnosis in a minority of cases. The development of affordable alternative molecular techniques may help with these histologically unresolved cases.

Global and single-nucleotide resolution detection of 7-methylguanosine in RNA.

RNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.

Molecular detection of exosomal miRNAs of blood serum for prognosis of colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer affecting people. The discovery of new, non-invasive, specific, and sensitive molecular biomarkers for CRC may assist in the diagnosis and support therapeutic decision making. Exosomal miRNAs have been demonstrated in carcinogenesis and CRC development, which makes these miRNAs strong biomarkers for CRC. Deep sequencing allows a robust high-throughput informatics investigation of the types and abundance of exosomal miRNAs. Thus, exosomal miRNAs can be efficiently examined as diagnostic biomarkers for disease screening. In the present study, a number of 660 mature miRNAs were detected in patients diagnosed with CRC at different stages. Of which, 29 miRNAs were differentially expressed in CRC patients compared with healthy controls. Twenty-nine miRNAs with high abundance levels were further selected for subsequent analysis. These miRNAs were either highly up-regulated (e.g., let-7a-5p, let-7c-5p, let-7f-5p, let-7d-3p, miR-423-5p, miR-3184-5p, and miR-584) or down-regulated (e.g., miR-30a-5p, miR-99-5p, miR-150-5p, miR-26-5p and miR-204-5p). These miRNAs influence critical genes in CRC, leading to either tumor growth or suppression. Most of the reported diagnostic exosomal miRNAs were shown to be circulating in blood serum. The latter is a novel miRNA that was found in exosomal profile of blood serum. Some of the predicted target genes of highly expressed miRNAs participate in several cancer pathways, including CRC pathway. These target genes include tumor suppressor genes, oncogenes and DNA repair genes. Main focus was given to multiple critical signaling cross-talking pathways including transforming growth factor ß (TGFß) signaling pathways that are directly linked to CRC. In conclusion, we recommend further analysis in order to experimentally confirm exact relationships between selected differentially expressed miRNAs and their predicted target genes and downstream functional consequences.

The use of next-generation sequencing in personalized medicine.

The revolutionary progress in development of next-generation sequencing (NGS) technologies has made it possible to deliver accurate genomic information in a timely manner. Over the past several years, NGS has transformed biomedical and clinical research and found its application in the field of personalized medicine. Here we discuss the rise of personalized medicine and the history of NGS. We discuss current applications and uses of NGS in medicine, including infectious diseases, oncology, genomic medicine, and dermatology. We provide a brief discussion of selected studies where NGS was used to respond to wide variety of questions in biomedical research and clinical medicine. Finally, we discuss the challenges of implementing NGS into routine clinical use.

Improved detection of low-frequency within-host variants from deep sequencing: A case study with human papillomavirus.

High-coverage sequencing allows the study of variants occurring at low frequencies within samples, but is susceptible to false-positives caused by sequencing error. Ion Torrent has a very low single nucleotide variant (SNV) error rate and has been employed for the majority of human papillomavirus (HPV) whole genome sequences. However, benchmarking of intrahost SNVs (iSNVs) has been challenging, partly due to limitations imposed by the HPV life cycle. We address this problem by deep sequencing three replicates for each of 31 samples of HPV type 18 (HPV18). Errors, defined as iSNVs observed in only one of three replicates, are dominated by C→T (G→A) changes, independently of trinucleotide context. True iSNVs, defined as those observed in all three replicates, instead show a more diverse SNV type distribution, with particularly elevated C→T rates in CCG context (CCG→CTG; CGG→CAG) and C→A rates in ACG context (ACG→AAG; CGT→CTT). Characterization of true iSNVs allowed us to develop two methods for detecting true variants: (1) VCFgenie, a dynamic binomial filtering tool which uses each variant's allele count and coverage instead of fixed frequency cut-offs; and (2) a machine learning binary classifier which trains eXtreme Gradient Boosting models on variant features such as quality and trinucleotide context. Each approach outperforms fixed-cut-off filtering of iSNVs, and performance is enhanced when both are used together. Our results provide improved methods for identifying true iSNVs in within-host applications across sequencing platforms, specifically using HPV18 as a case study.

Identification of a lncRNA/circRNA-miRNA-mRNA network in Nasopharyngeal Carcinoma by deep sequencing and bioinformatics analysis.

Background: Accumulating evidence indicates that non-coding RNAs (ncRNA), including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), can function as competitive endogenous RNAs (ceRNAs) by binding to microRNAs (miRNAs) and regulating host gene expression at the transcriptional or post-transcriptional level. Dysregulation in ceRNA network regulation has been implicated in the occurrence and development of cancer. However, the lncRNA/circRNA-miRNA-mRNA regulatory network is still lacking in nasopharyngeal carcinoma (NPC). Methods: Differentially expressed genes (DEGs) were obtained from our previous sequencing data and Gene Expression Omnibus (GEO). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) were used to explore the biological functions of these common DEGs. Through a series of bioinformatic analyses, the lncRNA/circRNA-miRNA-mRNA network was established. In additional, the external data GSE102349 was used to test the prognostic value of the hub mRNAs through the Kaplan-Meier method. Results: We successfully constructed a lncRNA/circRNA-miRNA-mRNA network in NPC, consisting of 16 lncRNAs, 6 miRNAs, 3 circRNAs and 10 mRNAs and found that three genes (TOP2A, ZWINT, TTK) were significantly associated with overall survival time (OS) in patients. Conclusion: The regulatory network revealed in this study may help comprehensively elucidate the ceRNA mechanisms driving NPC, and provide novel candidate biomarkers for evaluating the prognosis of NPC.

Rare subclonal sequencing of breast cancers indicates putative metastatic driver mutations are predominately acquired after dissemination.

BACKGROUND: Evolutionary models of breast cancer progression differ on the extent to which metastatic potential is pre-encoded within primary tumors. Although metastatic recurrences often harbor putative driver mutations that are not detected in their antecedent primary tumor using standard sequencing technologies, whether these mutations were acquired before or after dissemination remains unclear. METHODS: To ascertain whether putative metastatic driver mutations initially deemed specific to the metastasis by whole exome sequencing were, in actuality, present within rare ancestral subclones of the primary tumors from which they arose, we employed error-controlled ultra-deep sequencing (UDS-UMI) coupled with FFPE artifact mitigation by uracil-DNA glycosylase (UDG) to assess the presence of 132 "metastasis-specific" mutations within antecedent primary tumors from 21 patients. Maximum mutation detection sensitivity was ~1% of primary tumor cells. A conceptual framework was developed to estimate relative likelihoods of alternative models of mutation acquisition. RESULTS: The ancestral primary tumor subclone responsible for seeding the metastasis was identified in 29% of patients, implicating several putative drivers in metastatic seeding including LRP5 A65V and PEAK1 K140Q. Despite this, 93% of metastasis-specific mutations in putative metastatic driver genes remained undetected within primary tumors, as did 96% of metastasis-specific mutations in known breast cancer drivers, including ERRB2 V777L, ESR1 D538G, and AKT1 D323H. Strikingly, even in those cases in which the rare ancestral subclone was identified, 87% of metastasis-specific putative driver mutations remained undetected. Modeling indicated that the sequential acquisition of multiple metastasis-specific driver or passenger mutations within the same rare subclonal lineage of the primary tumor was highly improbable. CONCLUSIONS: Our results strongly suggest that metastatic driver mutations are sequentially acquired and selected within the same clonal lineage both before, but more commonly after, dissemination from the primary tumor, and that these mutations are biologically consequential. Despite inherent limitations in sampling archival primary tumors, our findings indicate that tumor cells in most patients continue to undergo clinically relevant genomic evolution after their dissemination from the primary tumor. This provides further evidence that metastatic recurrence is a multi-step, mutation-driven process that extends beyond primary tumor dissemination and underscores the importance of longitudinal tumor assessment to help guide clinical decisions.

Complete genome sequence of a novel papillomavirus in Siamese fighting fish (Betta splendens) from Trinidad and Tobago.

Here, we announce the complete genome of a previously undescribed papillomavirus from a betta fish, Betta splendens. The genome is 5,671 bp with a GC content of 38.2%. Variants were detected in public databases. This genome is most similar to papillomaviruses that infect sea bass (52.9 % nucleotide identity).

N6-methyladenosine in 5' UTR does not promote translation initiation.

The most abundant N6-methyladenosine (m6A) modification on mRNAs is installed non-stoichiometrically across transcripts, with 5' untranslated regions (5' UTRs) being the least conductive. 5' UTRs are essential for translation initiation, yet the molecular mechanisms orchestrated by m6A remain poorly understood. Here, we combined structural, biochemical, and single-molecule approaches and show that at the most common position, a single m6A does not affect translation yields, the kinetics of translation initiation complex assembly, or start codon recognition both under permissive growth and following exposure to oxidative stress. Cryoelectron microscopy (cryo-EM) structures of the late preinitiation complex reveal that m6A purine ring established stacking interactions with an arginine side chain of the initiation factor eIF2α, although with only a marginal energy contribution, as estimated computationally. These findings provide molecular insights into m6A interactions with the initiation complex and suggest that the subtle stabilization is unlikely to affect the translation dynamics under homeostatic conditions or stress.

Study of serum exosome miRNA as a biomarker for early onset adult ouclar myastthenia gravis.

BACKGROUND: By extracting and sequencing miRNAs from serum exosomes of patients with early-onset ocular myasthenia gravis (OMG), generalized myasthenia gravis (GMG) and healthy controls, we screened differentially expressed miRNAs and explored the possibility as potential biomarkers for early-onset OMG. METHODS: Peripheral blood samples were collected from patients with early-onset OMG, early-onset GMG, and age-matched healthy subjects, with 6 samples in each group. All these patients were diagnosed as MG for the first time and did not undergo any treatment. Exosomes miRNAs were extracted from the serum and performed deep sequencing; the differentially expressed miRNAs were compared and analyzed between OMG, GMG, and healthy control groups using edgeR. The differential expression standard was set to | log2FC |>1, p < 0.05. Target prediction of mRNAs were performed using miRTarBase, TargetScan, and miRDB databases, and a protein-protein interaction (PPI) network was constructed subsequently. The miRNAs with a significant difference were validated using RT-qPCR (10 early-onset OMG patients, 10 early-onset GMG patients and 10 age-sex-matched healthy subjects), and the value of the area under the ROC curve (AUC) was used to assess the diagnostic accuracy and evaluate clinical prognostic value. RESULTS: In total, one upregulated (miR-130a-3p) miRNA was obtained through the upregulated intersection between control vs OMG and OMG vs GMG; four downregulated (miR-4712-3p; miR-6752-5p; miR-320d; miR-3614-3p) miRNAs were obtained through the downregulated intersection between control vs OMG and OMG vs GMG. A total of 408 target genes were predicted for the five differentially expressed miRNAs. The mTOR signaling pathway and Rap1 signaling pathway were significantly enriched based on the enrichment results. RT-qPCR findings revealed that for the OMG, the expression of miR-320d, miR-4712-3p and miR-3614-3p was markedly up-/down-regulated as compared to GMG and healthy control group. The AUC for the three miRNAs between OMG and healthy control groups were 0.78, 0.79 and 0.79 respectively; the AUC between OMG and GMG was 0.84. CONCLUSIONS: The present study identified three novel miRNAs as candidate biomarkers for early-onset OMG patients and it was expected to provide a possibility and a new orientation for serum exosomal miRNAs as OMG diagnostic biomarkers.

Puzzles, challenges, and information reservoir of SARS-CoV-2 quasispecies.

Upon the emergence of SARS-CoV-2 in the human population, it was conjectured that for this coronavirus the dynamic intra-host heterogeneity typical of RNA viruses would be toned down. Nothing of this sort is observed. Here we review the main observations on the complexity and diverse composition of SARS-CoV-2 mutant spectra sampled from infected patients, within the framework of quasispecies dynamics. The analyses suggest that the information provided by myriads of genomic sequences within infected individuals may have a predictive value of the genomic sequences that acquire epidemiological relevance. Possibilities to reconcile the presence of broad mutant spectra in the large RNA coronavirus genome with its encoding a 3' to 5' exonuclease proofreading-repair activity are considered. Indeterminations in the behavior of individual viral genomes provide a benefit for the survival of the ensemble. We propose that this concept falls in the domain of "stochastic thinking," a notion that applies also to cellular processes, as a means for biological systems to face unexpected needs.

Impact of CRISPR/Cas9-Mediated CD73 Knockout in Pancreatic Cancer.

Pancreatic cancer is among the cancers with the highest mortality rates. Most of the patients are found to have advanced cancer, losing the chance of surgical treatment, and there is an urgent need to find new treatment methods. Targeted therapy for specific genes that play a key role in cancer is now an important means to improve the survival rate of patients. We determined that CD73 is highly expressed in pancreatic cancer by flow cytometry and qRT-PCR assays combined with bioinformatics techniques. Application of CRISPR/Cas9 technology to knockout CD73 in human and murine cell lines, respectively, revealed that CD73 inactivation inhibited cell growth and migration and induced G1 cell cycle arrest. We also found that CD73 deletion inhibited the ERK/STAT3 pathway and activated the E-cadherin pathway. In addition, a CRISPR/Cas9 protein kinase library screen was performed and identified Pbk, Fastk, Cdk19, Adck5, Trim28, and Pfkp as possible genes regulating CD73.

Assessment of bacterial and viral gut communities in healthy and tumoral colorectal tissue using RNA and DNA deep sequencing.

BACKGROUND: Colorectal cancer (CRC) is known to present a distinct microbiome profile compared to healthy mucosa. Non-targeted deep-sequencing strategies enable nowadays full microbiome characterization up to species level. AIM: We aimed to analyze both bacterial and viral communities in CRC using these strategies. MATERIALS & METHODS: We analyzed bacterial and viral communities using both DNA and RNA deep-sequencing (Novaseq) in colorectal tissue specimens from 10 CRC patients and 10 matched control patients. Following taxonomy classification using Kraken 2, different metrics for alpha and beta diversities as well as relative and differential abundance were calculated to compare tumoral and healthy samples. RESULTS: No viral differences were identified between tissue types, but bacterial species Polynucleobacter necessarius had a highly increased presence for DNA in tumors (p = 0.001). RNA analyses showed that bacterial species Arabia massiliensis had a highly decreased transcription in tumors (p = 0.002) while Fusobacterium nucleatum transcription was highly increased in tumors (p = 0.002). DISCUSSION: Sequencing of both DNA and RNA enables a wider perspective of micriobiome profiles. Lack of RNA transcription (Polynucleobacter necessarius) casts doubt on possible role of a microorganism in CRC. The association of F. nucleatum mainly with transcription, may provide further insights on its role in CRC. CONCLUSION: Joint assessment of the metagenome (DNA) and the metatranscriptome (RNA) at the species level provided a huge coverage for both bacteria and virus and identifies differential specific bacterial species as tumor associated.

Localization of Insertion Sequences in Plasmids for L-Cysteine Production in E. coli.

Insertion sequence elements (ISE) are often found to be responsible for the collapse of production in synthetically engineered Escherichia coli. By the transposition of ISE into the open reading frame of the synthetic pathway, E. coli cells gain selection advantage over cells expressing the metabolic burdensome production genes. Here, we present the exact entry sites of insertion sequence (IS) families 3 and 5 within plasmids for l-cysteine production in evolved E. coli populations. Furthermore, we identified an uncommon occurrence of an 8-bp direct repeat of IS5 which is atypical for this particular family, potentially indicating a new IS5 target site.

Deep Sequencing and Molecular Characterisation of BK Virus and JC Virus WHO International Reference Materials for Clinical Diagnostic Use.

BACKGROUND: Reactivation of JC and BK polyomaviruses during immunosuppression can lead to adverse clinical outcomes. In renal transplant recipients, BKV-associated nephropathy can result in graft loss, while in patients with autoimmune disorders, prolonged immunomodulatory drug use can cause rare onset of progressive multifocal leukoencephalopathy due to JCV reactivation. In such patients, accurate BK and JC viral load determinations by molecular technologies are important for diagnosis and clinical management; however, comparability across centres requires effective standardisation of diagnostic molecular detection systems. In October 2015, the WHO Expert Committee for Biological Standardisation (ECBS) established the 1st WHO International Standards (ISs) for use as primary-order calibrants for BKV and JCV nucleic acid detection. Two multi-centre collaborative studies confirmed their utility in harmonising agreement across the wide range of BKV and JCV assays, respectively. Previous Illumina-based deep sequence analysis of these standards, however, identified deletions in different regions, including the large T-antigen coding region. Hence, further detailed characterization was warranted. METHODS: Comprehensive sequence characterisation of each preparation using short- and long-read next-generation sequencing technologies was performed with additional corroborative independent digital PCR (dPCR) determinations. Potential error rates associated with long-read sequencing were minimised by applying rolling circle amplification (RCA) protocols for viral DNA (circular dsDNA), generating a full validation of sequence identity and composition and delineating the integrity of full-length BK and JC genomes. RESULTS: The analysed genomes displayed subpopulations frequently characterised by complex gene re-arrangements, duplications and deletions. CONCLUSIONS: Despite the recognition of such polymorphisms using high-resolution sequencing methodologies, the ability of these reference materials to act to enhance assay harmonisation did not appear significantly impacted, based on data generated by the 2015 WHO collaborative studies, but highlights cautionary aspects of IS generation and commutability for clinical molecular diagnostic application.

Accurate Detection of Urothelial Bladder Cancer Using Targeted Deep Sequencing of Urine DNA.

Patients with hematuria are commonly given an invasive cystoscopy test to detect bladder cancer (BC). To avoid the risks associated with cystoscopy, several urine-based methods for BC detection have been developed, the most prominent of which is the deep sequencing of urine DNA. However, the current methods for urine-based BC detection have significant levels of false-positive signals. In this study, we report on uAL100, a method to precisely detect BC tumor DNA in the urine without tumor samples. Using urine samples from 43 patients with BC and 21 healthy donors, uAL100 detected BC with 83.7% sensitivity and 100% specificity. The mutations identified in the urine DNA by uAL100 for BC detection were highly associated with BC tumorigenesis and progression. We suggest that uAL100 has improved accuracy compared to other urine-based methods for early BC detection and can reduce unnecessary cystoscopy tests for patients with hematuria.

Directed Evolution of Seneca Valley Virus in Tumorsphere and Monolayer Cell Cultures of a Small-Cell Lung Cancer Model.

The Seneca Valley virus (SVV) is an oncolytic virus from the picornavirus family, characterized by a 7.3-kilobase RNA genome encoding for all the structural and functional viral proteins. Directed evolution by serial passaging has been employed for oncolytic virus adaptation to increase the killing efficacy towards certain types of tumors. We propagated the SVV in a small-cell lung cancer model under two culture conditions: conventional cell monolayer and tumorspheres, with the latter resembling more closely the cellular structure of the tumor of origin. We observed an increase of the virus-killing efficacy after ten passages in the tumorspheres. Deep sequencing analyses showed genomic changes in two SVV populations comprising 150 single nucleotides variants and 72 amino acid substitutions. Major differences observed in the tumorsphere-passaged virus population, compared to the cell monolayer, were identified in the conserved structural protein VP2 and in the highly variable P2 region, suggesting that the increase in the ability of the SVV to kill cells over time in the tumorspheres is acquired by capsid conservation and positively selecting mutations to counter the host innate immune responses.

Characterization of clonal immunoglobulin heavy V-D-J gene rearrangements in Chinese patients with chronic lymphocytic leukemia: Clinical features and molecular profiles.

Introduction: Several prognostic factors of chronic lymphocytic leukemia (CLL) have been identified, such as cytogenetic aberrations and recurrent gene mutations. B-cell receptor (BCR) signaling plays an important role in the tumorigenesis of CLL, and its clinical significance in predicting prognosis is also under study. Methods: Therefore, we assessed the already-known prognostic markers, immunoglobulin heavy chain (IGH) gene usage and the associations among these factors in 71 patients diagnosed with CLL in our center from October 2017 to March 2022. Sequencing of IGH gene rearrangements was performed using Sanger sequencing or IGH-based next-generation sequencing, and the results were further analyzed for distinct IGH/IGHD/IGHJ genes and the mutational status of the clonotypic IGHV (IGH variable) gene. Results: In summary, by analyzing the distribution of potential prognostic factors in CLL patients, we displayed a landscape of molecular profiles, confirmed the predictive value of recurrent genetic mutations and chromosome aberrations, and found that IGHJ3 was associated with favorable markers (mutated IGHV, trisomy 12), while IGHJ6 tended to correlate with unfavorable factors (unmutated IGHV, del17p). Discussion: These results provided an indication for IGH gene sequencing in predicting the prognosis of CLL.

Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation.

RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.