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Baliga N, Stankiewicz K, Valenzuela J, Turkarslan S, Wu WJ, Gomez-Campo K, Locatelli N, Conn T, Radice V, Parker K, Alderdice R, Bay L, Voolstra C, Barshis D, Baums I. Alternative splicing in a coral during heat stress acclimation and recovery. RESEARCH SQUARE 2025:rs.3.rs-6025431. [PMID: 40235473 PMCID: PMC11998799 DOI: 10.21203/rs.3.rs-6025431/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/17/2025]
Abstract
Climate change has caused drastic declines in corals. As sessile organisms, corals acclimate to environmental shifts through genome-wide changes in gene expression, epigenetic modifications, and alterations in microbiome composition. However, alternative splicing (AS), a conserved mechanism of stress response in many organisms, has been under-explored in corals. Using short-term acute thermal stress assays, we investigated patterns of AS in the scleractinian coral Acropora cervicornis during response to low (33°C), medium (35°C), and high (37°C) heat stress and subsequent overnight recovery. Our findings demonstrate reproducible dynamic shifts in AS of at least 40 percent of all genes during response to heat treatment and the recovery phase. The relative proportion of AS increased in response to heat stress and was primarily dominated by intron retention in specific classes of transcripts, including those related to splicing regulation itself. While AS returned to baseline levels post-exposure to low heat, AS persisted even after reprieve from higher levels of heat stress, which was associated with irreversible loss of photosynthetic efficiency of the symbiont. Our findings demonstrate that, although animals, corals are more plant-like in their likely usage of AS for regulating thermal stress response and recovery.
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You Q, Liu J, Zhang R, Wang Z, Zhang B, Guo W, Xu N, Bottillo I, Shao L. Splicing Analysis of Exonic TSC1 and TSC2 Gene Variants Causing Tuberous Sclerosis Complex. Hum Mutat 2025; 2025:1497712. [PMID: 40226305 PMCID: PMC11978479 DOI: 10.1155/humu/1497712] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2024] [Accepted: 03/17/2025] [Indexed: 04/15/2025]
Abstract
Tuberous sclerosis complex (TSC) is characterized by abnormalities in cell proliferation and migration, leading to the development of hamartomas, benign tumors, or malignant cancers, affecting both the skin and brain, as well as potentially impacting the heart, kidneys, lungs, and eyes, with varying patterns of involvement over a lifetime. It is primarily caused by mutations in the TSC1 and TSC2 genes. Aberrant splicing is a crucial factor in hereditary diseases. Alternative splicing is a key mechanism for expanding the diversity of the human proteome. Mutations disrupting canonical splice sites or splicing regulatory elements impede the utilization of splice sites, leading to exon skipping and intron retention. We comprehensively analyzed missense and nonsense mutations of TSC1 and TSC2 genes using bioinformatics tools and identified 10 candidate mutations affecting pre-mRNA splicing through minigene analysis. Mutations in TSC genes can lead to partial or complete exon skipping and/or intron retention through complex mechanisms. This study emphasizes the importance of evaluating their roles in the splicing of suspected pathogenic variants in TSC.
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Affiliation(s)
- Qingqing You
- Department of Nephrology, Qingdao Municipal Hospital (Group), Qingdao Hospital of University of Health and Rehabilitation Sciences, Qingdao, China
| | - Jingwei Liu
- Department of Cardiac Surgery, Qingdao Municipal Hospital (Group), Qingdao Hospital of University of Health and Rehabilitation Sciences, Qingdao, China
| | - Ran Zhang
- Department of Nephrology, Qingdao Municipal Hospital (Group), Qingdao Hospital of University of Health and Rehabilitation Sciences, Qingdao, China
| | - Zhi Wang
- School of Clinical Medicine, Shandong Second Medical University, Weifang, China
| | - Bingying Zhang
- School of Clinical Medicine, Shandong Second Medical University, Weifang, China
| | - Wencong Guo
- Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China
| | - Ning Xu
- Department of Nephrology, Qingdao Municipal Hospital (Group), Qingdao Hospital of University of Health and Rehabilitation Sciences, Qingdao, China
| | - Irene Bottillo
- Division of Medical Genetics, Department of Experimental Medicine, San Camillo-Forlanini Hospital, Sapienza University, Rome, Italy
| | - Leping Shao
- Department of Nephrology, (Fujian Provincial Clinical Research Center for Glomerular Nephritis), The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
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3
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Peretto L, D'angiolillo C, Ferraresi P, Balestra D, Pinotti M. Rescue of a panel of Hemophilia A-causing 5'ss splicing mutations by unique Exon-specific U1snRNA variants. Mol Med 2025; 31:121. [PMID: 40148820 PMCID: PMC11948882 DOI: 10.1186/s10020-025-01176-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2024] [Accepted: 03/18/2025] [Indexed: 03/29/2025] Open
Abstract
BACKGROUND Aberrant mRNA splicing is a well-established pathogenic mechanism for human disease, but its real impact is hardly predictable and underestimated. Splicing can be therefore modulated for therapeutic purposes, and splicing-switching molecules are in clinics for some diseases. Here, conscious that over 10% of all pathogenic mutations occurs at 5'ss, we aimed at characterizing and rescuing nine 5'ss mutations in three models of defective F8 exons whose skipping would lead to factor VIII (FVIII) deficiency (Hemophilia A), the most frequent coagulation factor disorder. METHODS HEK293T cells were transfected with F8 minigene variants, alone or with engineered U1 small nuclear RNAs (U1snRNAs), and splicing patterns analysed via RT-PCR. RESULTS All 5'ss mutations induced exon skipping, and the proportion of correct transcripts, not predictable by computational analysis, was consistent with residual FVIII levels in patients. For each exon we identified a unique engineered U1snRNAs, either compensatory or Exon Specific (ExSpeU1), able to rescue all mutations. Overall, ExSpeU1s were more effective than compensatory U1snRNAs, particularly in the defective exons 6 and 22. CONCLUSIONS Data highlight the importance of splicing assays to elucidate genotype-phenotype relationships and proved the correction efficacy of ExSpeU1s for each targeted defective F8 exon, thus expanding their translational potential for HA.
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Affiliation(s)
- Laura Peretto
- Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, 44121, Italy
| | - Claudia D'angiolillo
- Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, 44121, Italy
| | - Paolo Ferraresi
- Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, 44121, Italy
| | - Dario Balestra
- Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, 44121, Italy.
| | - Mirko Pinotti
- Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, 44121, Italy
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Lord J, Oquendo CJ, Wai HA, Douglas AGL, Bunyan DJ, Wang Y, Hu Z, Zeng Z, Danis D, Katsonis P, Williams A, Lichtarge O, Chang Y, Bagnall RD, Mount SM, Matthiasardottir B, Lin C, Hansen TVO, Leman R, Martins A, Houdayer C, Krieger S, Bakolitsa C, Peng Y, Kamandula A, Radivojac P, Baralle D. Predicting the impact of rare variants on RNA splicing in CAGI6. Hum Genet 2025; 144:243-251. [PMID: 38170232 PMCID: PMC11976748 DOI: 10.1007/s00439-023-02624-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2023] [Accepted: 11/18/2023] [Indexed: 01/05/2024]
Abstract
Variants which disrupt splicing are a frequent cause of rare disease that have been under-ascertained clinically. Accurate and efficient methods to predict a variant's impact on splicing are needed to interpret the growing number of variants of unknown significance (VUS) identified by exome and genome sequencing. Here, we present the results of the CAGI6 Splicing VUS challenge, which invited predictions of the splicing impact of 56 variants ascertained clinically and functionally validated to determine splicing impact. The performance of 12 prediction methods, along with SpliceAI and CADD, was compared on the 56 functionally validated variants. The maximum accuracy achieved was 82% from two different approaches, one weighting SpliceAI scores by minor allele frequency, and one applying the recently published Splicing Prediction Pipeline (SPiP). SPiP performed optimally in terms of sensitivity, while an ensemble method combining multiple prediction tools and information from databases exceeded all others for specificity. Several challenge methods equalled or exceeded the performance of SpliceAI, with ultimate choice of prediction method likely to depend on experimental or clinical aims. One quarter of the variants were incorrectly predicted by at least 50% of the methods, highlighting the need for further improvements to splicing prediction methods for successful clinical application.
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Affiliation(s)
- Jenny Lord
- Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK
| | | | - Htoo A Wai
- Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK
| | - Andrew G L Douglas
- Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK
- Oxford Centre for Genomic Medicine, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
| | - David J Bunyan
- Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK
- Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, UK
| | - Yaqiong Wang
- Center for Molecular Medicine, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai, 201102, China
| | - Zhiqiang Hu
- University of California, Berkeley, Berkeley, CA, 94720, USA
| | - Zishuo Zeng
- Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, 08873, USA
| | - Daniel Danis
- The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT, 06032, USA
| | - Panagiotis Katsonis
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Amanda Williams
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Olivier Lichtarge
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Yuchen Chang
- Agnes Ginges Centre for Molecular Cardiology at Centenary Institute, University of Sydney, Sydney, Australia
- Faculty of Medicine and Health, University of Sydney, Sydney, Australia
| | - Richard D Bagnall
- Agnes Ginges Centre for Molecular Cardiology at Centenary Institute, University of Sydney, Sydney, Australia
- Faculty of Medicine and Health, University of Sydney, Sydney, Australia
| | - Stephen M Mount
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA
| | - Brynja Matthiasardottir
- Graduate Program in Biological Sciences and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA
- Inflammatory Disease Section, National Human Genome Research Institute, Bethesda, MD, USA
| | | | - Thomas van Overeem Hansen
- Department of Clinical Genetics, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
- Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Raphael Leman
- Laboratoire de Biologie et Génétique du Cancer, Centre François Baclesse, Caen, France
- Inserm U1245, Cancer Brain and Genomics, Normandie Université, UNICAEN, FHU G4 génomique, Rouen, France
| | - Alexandra Martins
- Inserm U1245, Cancer Brain and Genomics, Normandie Université, UNIROUEN, FHU G4 génomique, Rouen, France
| | - Claude Houdayer
- Inserm U1245, Cancer Brain and Genomics, Normandie Université, UNIROUEN, FHU G4 génomique, Rouen, France
- Department of Genetics, Univ Rouen Normandie, INSERM U1245, FHU-G4 Génomique and CHU Rouen, 76000, Rouen, France
| | - Sophie Krieger
- Laboratoire de Biologie et Génétique du Cancer, Centre François Baclesse, Caen, France
- Inserm U1245, Cancer Brain and Genomics, Normandie Université, UNICAEN, FHU G4 génomique, Rouen, France
| | | | - Yisu Peng
- Khoury College of Computer Sciences, Northeastern University, Boston, MA, 02115, USA
| | - Akash Kamandula
- Khoury College of Computer Sciences, Northeastern University, Boston, MA, 02115, USA
| | - Predrag Radivojac
- Khoury College of Computer Sciences, Northeastern University, Boston, MA, 02115, USA
| | - Diana Baralle
- Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK.
- Wessex Clinical Genetics Service, University Hospital Southampton NHS Foundation Trust, Southampton, UK.
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Tan H, Gotea V, Jaiswal SK, Seidel NE, Holland DO, Fedkenheuer K, Elkahloun AG, Bang-Christensen SR, Elnitski L. iSoMAs: Finding isoform expression and somatic mutation associations in human cancers. PLoS Comput Biol 2025; 21:e1012847. [PMID: 40053523 PMCID: PMC12052144 DOI: 10.1371/journal.pcbi.1012847] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Revised: 05/05/2025] [Accepted: 02/03/2025] [Indexed: 03/09/2025] Open
Abstract
Aberrant alternative splicing, prevalent in cancer, impacts various cancer hallmarks involving proliferation, angiogenesis, and invasion. Splicing disruption often results from somatic point mutations rewiring functional pathways to support cancer cell survival. We introduce iSoMAs (iSoform expression and somatic Mutation Association), an efficient computational pipeline leveraging principal component analysis technique, to explore how somatic mutations influence transcriptome-wide gene expression at the isoform level. Applying iSoMAs to 33 cancer types comprising 9,738 tumor samples in The Cancer Genome Atlas, we identified 908 somatically mutated genes significantly associated with altered isoform expression across three or more cancer types. Mutations linked to differential isoform expression occurred through both cis- and trans-acting mechanisms, involving well-known oncogenes/suppressor genes, RNA binding protein and splicing factor genes. With wet-lab experiments, we verified direct association between TP53 mutations and differential isoform expression in cell cycle genes. Additional iSoMAs genes have been validated in the literature with independent cohorts and/or methods. Despite the complexity of cancer, iSoMAs attains computational efficiency via dimension reduction strategy and reveals critical associations between regulatory factors and transcriptional landscapes.
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Affiliation(s)
- Hua Tan
- Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Valer Gotea
- Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Sushil K. Jaiswal
- Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Nancy E. Seidel
- Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - David O. Holland
- Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Kevin Fedkenheuer
- Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Abdel G. Elkahloun
- Microarrays and Single-Cell Genomics Core, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Sara R. Bang-Christensen
- Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Laura Elnitski
- Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
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6
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Bak-Gordon P, Manley JL. SF3B1: from core splicing factor to oncogenic driver. RNA (NEW YORK, N.Y.) 2025; 31:314-332. [PMID: 39773890 PMCID: PMC11874996 DOI: 10.1261/rna.080368.124] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Accepted: 12/23/2024] [Indexed: 01/11/2025]
Abstract
Highly recurrent somatic mutations in the gene encoding the core splicing factor SF3B1 are drivers of multiple cancer types. SF3B1 is a scaffold protein that orchestrates multivalent protein-protein interactions within the spliceosome that are essential for recognizing the branchsite (BS) and selecting the 3' splice site during the earliest stage of pre-mRNA splicing. In this review, we first describe the molecular mechanism by which multiple oncogenic SF3B1 mutations disrupt splicing. This involves perturbation of an early spliceosomal trimeric protein complex necessary for accurate BS recognition in a subset of introns, which leads to activation of upstream branchpoints and selection of cryptic 3' splice sites. We next discuss how specific transcripts affected by aberrant splicing in SF3B1-mutant cells contribute to the initiation and progression of cancer. Finally, we highlight the prognostic value and disease phenotypes of different cancer-associated SF3B1 mutations, which is critical for developing new targeted therapeutics against SF3B1-mutant cancers still lacking in the clinic.
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Affiliation(s)
- Pedro Bak-Gordon
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| | - James L Manley
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
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7
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Miguel Berenguel L, Gianelli C, Matas Pérez E, del Rosal T, Méndez Echevarría A, Robles Marhuenda Á, Feito Rodríguez M, Caballero Molina MT, Magallares García L, Sánchez Garrido B, Hita Díaz S, Allende Martínez L, Nozal Aranda P, Cámara Hijón C, López Granados E, Rodríguez Pena R, Bravo García-Morato M. Molecular assessment of splicing variants in a cohort of patients with inborn errors of immunity: methodological approach and interpretation remarks. Front Immunol 2025; 15:1499415. [PMID: 39944559 PMCID: PMC11814461 DOI: 10.3389/fimmu.2024.1499415] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2024] [Accepted: 12/27/2024] [Indexed: 05/09/2025] Open
Abstract
Background Splicing is the molecular mechanism to produce mature messenger RNA (mRNA) before its translation into protein. It is estimated that 50% of disease-causing mutations disrupt splicing, mostly of them affecting canonical positions. However, variants occurring in coding regions or deep-intronic variants can also affect splicing. In these cases, interpretation of the results may be challenging and molecular validation is required. Methods The study includes 23 patients with splicing variants out of a cohort of 187 patients diagnosed with inborn errors of immunity (IEI). Clinical features and immunophenotypes are shown. Reverse transcription-polymerase chain reaction (RT-PCR) is the molecular assay employed for pathogenicity validation. Results We detected 23 patients of 20 pedigrees with splicing variants in IEI genes, which constitutes the 12.3% of our cohort. In total, 21 splicing variants were analyzed, 10 of which had previously been reported in the literature and 11 novel ones. Among the 23 patients, 16 showed variants at canonical splice sites. Molecular validation was required only in the cases of genes of uncertain significance (GUS), high homology pseudogenes or incompatible clinical phenotype. Seven patients showed variants outside canonical positions. All of them needed molecular validation, with the exception of two patients, whose variants had previously been well characterized in the medical literature. Conclusion This study shows the proportion of splicing variants in a cohort of IEI patients, providing their clinical phenotypic characteristics and the methodology used to validate the splicing defects. Based on the results, an algorithm is proposed to clarify when a splicing variant should be validated by complementary methodology and when, by contrast, it can be directly considered disease causing.
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Affiliation(s)
| | - Carla Gianelli
- Department of Immunology, La Paz University Hospital, Madrid, Spain
- Center for Biomedical Network Research on Rare Diseases (CIBERER U767), Madrid, Spain
- Lymphocyte Pathophysiology in Immunodeficiencies Group, La Paz Institute of Biomedical Research, Madrid, Spain
| | | | - Teresa del Rosal
- Department of Pediatric Infectious Diseases, La Paz University Hospital, Madrid, Spain
| | - Ana Méndez Echevarría
- Department of Pediatric Infectious Diseases, La Paz University Hospital, Madrid, Spain
| | | | | | - Maria Teresa Caballero Molina
- Department of Allergy, La Paz University Hospital, Madrid, Spain
- Center for Biomedical Network Research on Rare Diseases (CIBERER U754), Madrid, Spain
| | | | | | | | - Luis Allende Martínez
- Immunology Department, 12 de Octubre University Hospital, Madrid, Spain
- Research Institute Hospital 12 Octubre (I+12), Madrid, Spain
| | - Pilar Nozal Aranda
- Department of Immunology, La Paz University Hospital, Madrid, Spain
- Center for Biomedical Network Research on Rare Diseases (CIBERER U754), Madrid, Spain
- Complement Alterations in Human Pathology Group, La Paz Institute of Biomedical Research, Madrid, Spain
| | - Carmen Cámara Hijón
- Department of Immunology, La Paz University Hospital, Madrid, Spain
- Center for Biomedical Network Research on Rare Diseases (CIBERER U767), Madrid, Spain
- Lymphocyte Pathophysiology in Immunodeficiencies Group, La Paz Institute of Biomedical Research, Madrid, Spain
| | - Eduardo López Granados
- Department of Immunology, La Paz University Hospital, Madrid, Spain
- Center for Biomedical Network Research on Rare Diseases (CIBERER U767), Madrid, Spain
- Lymphocyte Pathophysiology in Immunodeficiencies Group, La Paz Institute of Biomedical Research, Madrid, Spain
| | - Rebeca Rodríguez Pena
- Department of Immunology, La Paz University Hospital, Madrid, Spain
- Center for Biomedical Network Research on Rare Diseases (CIBERER U767), Madrid, Spain
- Lymphocyte Pathophysiology in Immunodeficiencies Group, La Paz Institute of Biomedical Research, Madrid, Spain
| | - María Bravo García-Morato
- Department of Immunology, La Paz University Hospital, Madrid, Spain
- Center for Biomedical Network Research on Rare Diseases (CIBERER U767), Madrid, Spain
- Lymphocyte Pathophysiology in Immunodeficiencies Group, La Paz Institute of Biomedical Research, Madrid, Spain
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8
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Zheng H, Cheng C, He M, Zhou W, Li Y, Dai J, Zhang T, Xu K, Zhang X, Tian X, Liu Y. Interpreting Variants of Uncertain Significance in PCD: Abnormal Splicing Caused by a Missense Variant of DNAAF3. Mol Genet Genomic Med 2025; 13:e70036. [PMID: 39764684 PMCID: PMC11705539 DOI: 10.1002/mgg3.70036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2024] [Revised: 08/16/2024] [Accepted: 11/06/2024] [Indexed: 01/11/2025] Open
Abstract
BACKGROUND Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by dysfunction of motile cilia. While approximately 50 genes have been identified, around 25% of PCD patients remain genetically unexplained; elucidating the pathogenicity of specific variants remains a challenge. METHODS Whole exome sequencing (WES) and Sanger sequencing were conducted to identify potential pathogenic variants of PCD. Minigene assays were performed to evaluate the pathogenicity of variants. Transmission electron microscopy (TEM) and high-speed video analysis (HSVA) were conducted to analyze the function of cilia in respiratory epithelial cells. RESULTS We identified two variants of DNAAF3: c.557G>A, p.G186E in exon 5, and c.1364G>A, p.G455D at the terminal nucleotide of exon 10 in a 16-year-old male patient. Through a minigene assay, we demonstrated that the c.1364G>A variant led to a four-nucleotide skipping. The cilia in epithelial ciliary cells of the proband were almost immotile. The absence of outer dynein arms and inner dynein arms was also observed. CONCLUSIONS Our study identified two compound heterozygous variants of DNAAF3, a pathogenic gene for PCD, and proved that a novel missense variant c.1364G>A affects splicing. Our findings not only expanded the spectrum of mutations in the DNAAF3 gene but also highlighted the importance of investigating variants of uncertain significance (VUS) for comprehensive genetic diagnoses.
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Affiliation(s)
- Haixia Zheng
- McKusick‐Zhang Center for Genetic Medicine, State Key Laboratory for Complex Severe and Rare Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical SciencesSchool of Basic Medicine, Peking Union Medical CollegeBeijingChina
| | - Chongsheng Cheng
- Department of Pulmonary and Critical Care Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College HospitalChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina
| | - Miao He
- McKusick‐Zhang Center for Genetic Medicine, State Key Laboratory for Complex Severe and Rare Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical SciencesSchool of Basic Medicine, Peking Union Medical CollegeBeijingChina
| | - Wangji Zhou
- Department of Pulmonary and Critical Care Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College HospitalChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina
| | - Yixuan Li
- McKusick‐Zhang Center for Genetic Medicine, State Key Laboratory for Complex Severe and Rare Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical SciencesSchool of Basic Medicine, Peking Union Medical CollegeBeijingChina
| | - Jinrong Dai
- Department of Pulmonary and Critical Care Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College HospitalChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina
| | - Ting Zhang
- Department of Pulmonary and Critical Care Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College HospitalChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina
| | - Kai‐Feng Xu
- Department of Pulmonary and Critical Care Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College HospitalChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina
| | - Xue Zhang
- McKusick‐Zhang Center for Genetic Medicine, State Key Laboratory for Complex Severe and Rare Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical SciencesSchool of Basic Medicine, Peking Union Medical CollegeBeijingChina
| | - Xinlun Tian
- Department of Pulmonary and Critical Care Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College HospitalChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina
| | - Yaping Liu
- The State Key Laboratory for Complex Severe and Rare Diseases, the State Key Sci‐Tech Infrastructure for Translational MedicinePeking Union Medical College HospitalBeijingChina
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9
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Duan C, Rong S, Buerer L, Neil CR, Savatt JM, Strande NT, Fairbrother WG. One-Size-Fits-Many: Antisense oligonucleotides for rescuing splicing mutations in hotspot exons. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.07.627366. [PMID: 39677675 PMCID: PMC11643266 DOI: 10.1101/2024.12.07.627366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2024]
Abstract
Mutations that impact splicing play a significant role in disease etiology but are not fully understood. To characterize the impact of exonic variants on splicing in 71 clinically-actionable disease genes in asymptomatic people, we analyzed 32,112 exonic mutations from ClinVar and Geisinger MyCode using a minigene reporter assay. We identify 1,733 splice-disrupting mutations, of which the most extreme 1-2% of variants are likely to be deleterious. We report that these variants are not distributed evenly across exons but are mostly concentrated in the ∼8% of exons that are most susceptible to splicing mutations (i.e. hotspot exons). We demonstrate that splicing defects in these exons can be reverted by ASOs targeting the splice sites of either their upstream or downstream flanking exons. This finding supports the feasibility of developing single therapeutic ASOs that could revert all splice-altering variants localized to a particular exon.
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10
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Ciccolella S, Cozzi D, Della Vedova G, Kuria SN, Bonizzoni P, Denti L. Differential quantification of alternative splicing events on spliced pangenome graphs. PLoS Comput Biol 2024; 20:e1012665. [PMID: 39652592 DOI: 10.1371/journal.pcbi.1012665] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2024] [Revised: 12/19/2024] [Accepted: 11/21/2024] [Indexed: 12/21/2024] Open
Abstract
Pangenomes are becoming a powerful framework to perform many bioinformatics analyses taking into account the genetic variability of a population, thus reducing the bias introduced by a single reference genome. With the wider diffusion of pangenomes, integrating genetic variability with transcriptome diversity is becoming a natural extension that demands specific methods for its exploration. In this work, we extend the notion of spliced pangenomes to that of annotated spliced pangenomes; this allows us to introduce a formal definition of Alternative Splicing (AS) events on a graph structure. To investigate the usage of graph pangenomes for the quantification of AS events across conditions, we developed pantas, the first pangenomic method for the detection and differential analysis of AS events from short RNA-Seq reads. A comparison with state-of-the-art linear reference-based approaches proves that pantas achieves competitive accuracy, making spliced pangenomes effective for conducting AS events quantification and opening future directions for the analysis of population-based transcriptomes.
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Affiliation(s)
- Simone Ciccolella
- Department of Computer Science, University of Milano-Bicocca, Milan, Italy
| | - Davide Cozzi
- Department of Computer Science, University of Milano-Bicocca, Milan, Italy
| | | | | | - Paola Bonizzoni
- Department of Computer Science, University of Milano-Bicocca, Milan, Italy
| | - Luca Denti
- Department of Computer Science, University of Milano-Bicocca, Milan, Italy
- Department of Applied Informatics, Faculty of Mathematics, Physics and Informatics, Comenius University in Bratislava, Bratislava, Slovakia
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11
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Zhu L, Chen M, Shi Y, Huang X, Ding H. Prenatal detection of novel compound heterozygous variants of the PLD1 gene in a fetus with congenital heart disease. Front Genet 2024; 15:1498485. [PMID: 39553471 PMCID: PMC11564120 DOI: 10.3389/fgene.2024.1498485] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2024] [Accepted: 10/18/2024] [Indexed: 11/19/2024] Open
Abstract
Background Congenital heart disease (CHD) is the most common birth defect and heart valve defects are the most common cardiac defect, accounting for over 25% of all congenital heart diseases. To date, more than 400 genes have been linked to CHD, the genetic analysis of CHD cases is crucial for both clinical management and etiological determination. Patients with autosomal-recessive variants of PLD1 are predisposed to Cardiac Valvular Dysplasia-1 (CVDP1), which predominantly affects the right-sided heart valves, including the pulmonic, tricuspid, and mitral valves. Methods Databases were utilized to predict the impact of the c.1062-59A>G variant on splicing. Whole-exome sequencing (WES), reverse transcription polymerase chain reaction (RT-PCR), Sanger sequencing, and TA clone sequencing were conducted on both the parents and the fetus. Results A compound heterozygous variation in the PLD1(NM_002662.5):c.1937G>C (p. G646A) from the father and PLD1(NM_002662.5):c.1062-59A>G from the mother, was identified and confirmed in the fetus. The c.1937G>C (p. G646A) and the c.1062-59A>G variants were all classified as variant of uncertain significance (VUS) per ACMG guidelines. RT-PCR and TA clone sequencing revealed a 76-bp intronic insertion and exon 11 skipping in the proband and her mother's transcripts, causing a frameshift and premature stop codon in PLD1. Consequently, after being informed about the risks of their variant of unknown significance (VUS), the couple chose pre-implantation genetic testing for monogenic disorders (PGT-M) and had a healthy child. Conclusion Our study identified novel variants to expand the mutation spectrum of CHD and provided reliable evidence for the recurrent risk and reproductive care options.
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Affiliation(s)
- Linyan Zhu
- Department of Obstetrics and Gynaecology, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China
- Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
| | - Mei Chen
- Department of Obstetrics and Gynaecology, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China
| | - Yubo Shi
- Department of Obstetrics and Gynaecology, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China
| | - Xiaxi Huang
- Department of Obstetrics and Gynaecology, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China
| | - Huiqing Ding
- Department of Obstetrics and Gynaecology, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China
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12
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Wang Y, Shu M, Wang T, He T, Yuan J, Yang Y. Comprehensive characterization of somatic mutations associated with chimeric RNAs in human cancers. Int J Cancer 2024; 155:683-696. [PMID: 38613405 DOI: 10.1002/ijc.34955] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2023] [Revised: 03/14/2024] [Accepted: 03/19/2024] [Indexed: 04/14/2024]
Abstract
Chimeric RNAs, which can arise from gene recombination at the DNA level or non-canonical splicing events at the RNA level, have been identified as important roles in human tumors. Dysregulated gene expression caused by somatic mutations and altered splicing patterns of oncogenes or tumor suppressor genes can contribute to the development of tumors. Therefore, investigating the formation mechanism of chimeric RNAs via somatic mutations is critical for understanding tumor pathogenesis. This project is the first to propose studying the association between somatic single nucleotide variants and chimeric RNAs, identifying around 2900 somatic SNVs affecting chimeric RNAs in pan-cancer level. The somatic SNVs on chimeric RNAs were commonly observed in various types of tumor tissues, providing a valuable resource for future study. Additionally, these SNVs show distinct tumor specificity, and those with high frequency had a significant impact on the survival time of patients with tumors. Further research revealed that somatic SNVs associated with chimeric RNA (chiR-SNVs) were typically found within 10 nt of the junction site of chimeric RNAs and had a particularly significant effect on chimeric RNAs from different chromosomes. The enrichment analysis revealed that chiR-SNVs were significantly overrepresented in oncogenes and genes related to RNA binding proteins involved in RNA splicing, which could imply that chiR-SNVs may disrupt the process of RNA splicing and induce the occurrence of chimeric RNAs. This study sheds light on the potential molecular interaction mechanism between somatic SNVs and chimeric RNAs, which opens up a new avenue for researching disease pathway and tumorigenesis development.
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Affiliation(s)
- Yuting Wang
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Key Laboratory of Inflammatory Biology, Tianjin Geriatrics Institute, Tianjin Medical University General Hospital, Department of Bioinformatics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
- Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
| | - Meng Shu
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Key Laboratory of Inflammatory Biology, Tianjin Geriatrics Institute, Tianjin Medical University General Hospital, Department of Bioinformatics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
- Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
| | - Tianqiang Wang
- Neurosurgery Department II Ward, Yidu Central Hospital of Weifang, Shandong, China
| | - Tongxin He
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Key Laboratory of Inflammatory Biology, Tianjin Geriatrics Institute, Tianjin Medical University General Hospital, Department of Bioinformatics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
| | - Jiapei Yuan
- State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China
| | - Yang Yang
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Key Laboratory of Inflammatory Biology, Tianjin Geriatrics Institute, Tianjin Medical University General Hospital, Department of Bioinformatics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
- Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
- Department of Family Planning, The Second Hospital of Tianjin Medical University, Tianjin, China
- Department of Geriatrics, Tianjin Medical University General Hospital, Tianjin Medical University, Tianjin, China
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13
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Vecoli C, Foffa I, Vittorini S, Botto N, Esposito A, Costa S, Piagneri V, Festa P, Ait-Ali L. A novel TGFβR2 splice variant in patient with aortic aneurysm and family history for aortic dissection: a case report. Per Med 2024; 21:139-144. [PMID: 38634413 DOI: 10.2217/pme-2023-0135] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Accepted: 03/26/2024] [Indexed: 04/19/2024]
Abstract
We report the clinical presentation and genetic screening of a 31-year-old man with dilatation of the aortic root and ascending aorta and a positive family history for aortic dissection and sudden death. A novel heterozygous variant in a splice acceptor site (c.1600-1G>T) of TGFβR2 gene was identified by using a targeted multi-gene panel analysis. Bioinformatics tools predicted that the c.1600-1G>T variant is pathogenic by altering acceptor splice site at - 1 position affecting pre-mRNA splicing. These data confirm that the diverging splicing in the TGF-β pathway genes may be an important process in aneurismal disease and emphasize the utility of genetic sequencing in the identification of high-risk patients for a more patient's management able to improve outcomes and minimize costs for the care of patients with heritable thoracic aortic aneurysm and dissection.
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Affiliation(s)
- Cecilia Vecoli
- Institute of Clinical Physiology, CNR, Massa, Italy
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
| | - Ilenia Foffa
- Institute of Clinical Physiology, CNR, Massa, Italy
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
| | - Simona Vittorini
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
| | - Nicoletta Botto
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
| | - Augusto Esposito
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
| | - Sabrina Costa
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
| | - Valeria Piagneri
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
| | - Pierluigi Festa
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
| | - Lamia Ait-Ali
- Institute of Clinical Physiology, CNR, Massa, Italy
- Ospedale del Cuore, Fondazione Toscana "G. Monasterio", Massa, Italy
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14
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Speakman E, Gunaratne GH. On a kneading theory for gene-splicing. CHAOS (WOODBURY, N.Y.) 2024; 34:043125. [PMID: 38579148 DOI: 10.1063/5.0199364] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/22/2024] [Accepted: 03/05/2024] [Indexed: 04/07/2024]
Abstract
Two well-known facets in protein synthesis in eukaryotic cells are transcription of DNA to pre-RNA in the nucleus and the translation of messenger-RNA (mRNA) to proteins in the cytoplasm. A critical intermediate step is the removal of segments (introns) containing ∼97% of the nucleic-acid sites in pre-RNA and sequential alignment of the retained segments (exons) to form mRNA through a process referred to as splicing. Alternative forms of splicing enrich the proteome while abnormal splicing can enhance the likelihood of a cell developing cancer or other diseases. Mechanisms for splicing and origins of splicing errors are only partially deciphered. Our goal is to determine if rules on splicing can be inferred from data analytics on nucleic-acid sequences. Toward that end, we represent a nucleic-acid site as a point in a plane defined in terms of the anterior and posterior sub-sequences of the site. The "point-set" representation expands analytical approaches, including the use of statistical tools, to characterize genome sequences. It is found that point-sets for exons and introns are visually different, and that the differences can be quantified using a family of generalized moments. We design a machine-learning algorithm that can recognize individual exons or introns with 91% accuracy. Point-set distributions and generalized moments are found to differ between organisms.
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Affiliation(s)
- Ethan Speakman
- Department of Physics, University of Houston, Houston, Texas 77204, USA
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15
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Chafai N, Bonizzi L, Botti S, Badaoui B. Emerging applications of machine learning in genomic medicine and healthcare. Crit Rev Clin Lab Sci 2024; 61:140-163. [PMID: 37815417 DOI: 10.1080/10408363.2023.2259466] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2023] [Accepted: 09/12/2023] [Indexed: 10/11/2023]
Abstract
The integration of artificial intelligence technologies has propelled the progress of clinical and genomic medicine in recent years. The significant increase in computing power has facilitated the ability of artificial intelligence models to analyze and extract features from extensive medical data and images, thereby contributing to the advancement of intelligent diagnostic tools. Artificial intelligence (AI) models have been utilized in the field of personalized medicine to integrate clinical data and genomic information of patients. This integration allows for the identification of customized treatment recommendations, ultimately leading to enhanced patient outcomes. Notwithstanding the notable advancements, the application of artificial intelligence (AI) in the field of medicine is impeded by various obstacles such as the limited availability of clinical and genomic data, the diversity of datasets, ethical implications, and the inconclusive interpretation of AI models' results. In this review, a comprehensive evaluation of multiple machine learning algorithms utilized in the fields of clinical and genomic medicine is conducted. Furthermore, we present an overview of the implementation of artificial intelligence (AI) in the fields of clinical medicine, drug discovery, and genomic medicine. Finally, a number of constraints pertaining to the implementation of artificial intelligence within the healthcare industry are examined.
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Affiliation(s)
- Narjice Chafai
- Laboratory of Biodiversity, Ecology, and Genome, Faculty of Sciences, Department of Biology, Mohammed V University in Rabat, Rabat, Morocco
| | - Luigi Bonizzi
- Department of Biomedical, Surgical and Dental Science, University of Milan, Milan, Italy
| | - Sara Botti
- PTP Science Park, Via Einstein - Loc. Cascina Codazza, Lodi, Italy
| | - Bouabid Badaoui
- Laboratory of Biodiversity, Ecology, and Genome, Faculty of Sciences, Department of Biology, Mohammed V University in Rabat, Rabat, Morocco
- African Sustainable Agriculture Research Institute (ASARI), Mohammed VI Polytechnic University (UM6P), Laâyoune, Morocco
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16
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Piron A, Szymczak F, Papadopoulou T, Alvelos MI, Defrance M, Lenaerts T, Eizirik DL, Cnop M. RedRibbon: A new rank-rank hypergeometric overlap for gene and transcript expression signatures. Life Sci Alliance 2024; 7:e202302203. [PMID: 38081640 PMCID: PMC10709657 DOI: 10.26508/lsa.202302203] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2023] [Revised: 11/28/2023] [Accepted: 11/29/2023] [Indexed: 12/18/2023] Open
Abstract
High-throughput omics technologies have generated a wealth of large protein, gene, and transcript datasets that have exacerbated the need for new methods to analyse and compare big datasets. Rank-rank hypergeometric overlap is an important threshold-free method to combine and visualize two ranked lists of P-values or fold-changes, usually from differential gene expression analyses. Here, we introduce a new rank-rank hypergeometric overlap-based method aimed at gene level and alternative splicing analyses at transcript or exon level, hitherto unreachable as transcript numbers are an order of magnitude larger than gene numbers. We tested the tool on synthetic and real datasets at gene and transcript levels to detect correlation and anticorrelation patterns and found it to be fast and accurate, even on very large datasets thanks to an evolutionary algorithm-based minimal P-value search. The tool comes with a ready-to-use permutation scheme allowing the computation of adjusted P-values at low time cost. The package compatibility mode is a drop-in replacement to previous packages. RedRibbon holds the promise to accurately extricate detailed information from large comparative analyses.
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Affiliation(s)
- Anthony Piron
- ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles, Brussels, Belgium
- Interuniversity Institute of Bioinformatics in Brussels (IB2), Brussels, Belgium
- Machine Learning Group, Université Libre de Bruxelles, Brussels, Belgium
| | - Florian Szymczak
- ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles, Brussels, Belgium
- Interuniversity Institute of Bioinformatics in Brussels (IB2), Brussels, Belgium
| | - Theodora Papadopoulou
- ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles, Brussels, Belgium
- Interuniversity Institute of Bioinformatics in Brussels (IB2), Brussels, Belgium
| | - Maria Inês Alvelos
- ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles, Brussels, Belgium
| | - Matthieu Defrance
- Interuniversity Institute of Bioinformatics in Brussels (IB2), Brussels, Belgium
- Machine Learning Group, Université Libre de Bruxelles, Brussels, Belgium
| | - Tom Lenaerts
- Interuniversity Institute of Bioinformatics in Brussels (IB2), Brussels, Belgium
- Machine Learning Group, Université Libre de Bruxelles, Brussels, Belgium
- Artificial Intelligence Lab, Vrije Universiteit Brussel, Brussels, Belgium
| | - Décio L Eizirik
- ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles, Brussels, Belgium
| | - Miriam Cnop
- ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles, Brussels, Belgium
- Division of Endocrinology, Erasmus Hospital, Université Libre de Bruxelles, Brussels, Belgium
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17
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Knill C, Henderson EJ, Johnson C, Wah VY, Cheng K, Forster AJ, Itasaki N. Defects of the spliceosomal gene SNRPB affect osteo- and chondro-differentiation. FEBS J 2024; 291:272-291. [PMID: 37584444 DOI: 10.1111/febs.16934] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Revised: 07/25/2023] [Accepted: 08/14/2023] [Indexed: 08/17/2023]
Abstract
Although gene splicing occurs throughout the body, the phenotype of spliceosomal defects is largely limited to specific tissues. Cerebro-costo-mandibular syndrome (CCMS) is one such spliceosomal disease, which presents as congenital skeletal dysmorphism and is caused by mutations of SNRPB gene encoding Small Nuclear Ribonucleoprotein Polypeptides B/B' (SmB/B'). This study employed in vitro cell cultures to monitor osteo- and chondro-differentiation and examined the role of SmB/B' in the differentiation process. We found that low levels of SmB/B' by knockdown or mutations of SNRPB led to suppressed osteodifferentiation in Saos-2 osteoprogenitor-like cells, which was accompanied by affected splicing of Dlx5. On the other hand, low SmB/B' led to promoted chondrogenesis in HEPM mesenchymal stem cells. Consistent with other reports, osteogenesis was promoted by the Wnt/β-catenin pathway activator and suppressed by Wnt and BMP blockers, whereas chondrogenesis was promoted by Wnt inhibitors. Suppressed osteogenic markers by SNRPB knockdown were partly rescued by Wnt/β-catenin pathway activation. Reporter analysis revealed that suppression of SNRPB results in attenuated Wnt pathway and/or enhanced BMP pathway activities. SNRPB knockdown altered splicing of TCF7L2 which impacts Wnt/β-catenin pathway activities. This work helps unravel the mechanism underlying CCMS whereby reduced expression of spliceosomal proteins causes skeletal phenotypes.
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Affiliation(s)
- Chris Knill
- Faculty of Life Sciences, University of Bristol, UK
| | | | - Craig Johnson
- Faculty of Health Sciences, University of Bristol, UK
| | - Vun Yee Wah
- Faculty of Life Sciences, University of Bristol, UK
| | - Kevin Cheng
- Faculty of Life Sciences, University of Bristol, UK
| | | | - Nobue Itasaki
- Faculty of Health Sciences, University of Bristol, UK
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18
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Li K, Xiao J, Ling Z, Luo T, Xiong J, Chen Q, Dong L, Wang Y, Wang X, Jiang Z, Xia L, Yu Z, Hua R, Guo R, Tang D, Lv M, Lian A, Li B, Zhao G, He X, Xia K, Cao Y, Li J. Prioritizing de novo potential non-canonical splicing variants in neurodevelopmental disorders. EBioMedicine 2024; 99:104928. [PMID: 38113761 PMCID: PMC10767160 DOI: 10.1016/j.ebiom.2023.104928] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2023] [Revised: 11/30/2023] [Accepted: 12/05/2023] [Indexed: 12/21/2023] Open
Abstract
BACKGROUND Genomic variants outside of the canonical splicing site (±2) may generate abnormal mRNA splicing, which are defined as non-canonical splicing variants (NCSVs). However, the clinical interpretation of NCSVs in neurodevelopmental disorders (NDDs) is largely unknown. METHODS We investigated the contribution of NCSVs to NDDs from 345,787 de novo variants (DNVs) in 47,574 patients with NDDs. We performed functional enrichment and protein-protein interaction analysis to assess the association between genes carrying prioritised NCSVs and NDDs. Minigene was used to validate the impact of NCSVs on mRNA splicing. FINDINGS We observed significantly more NCSVs (p = 0.02, odds ratio [OR] = 2.05) among patients with NDD than in controls. Both canonical splicing variants (CSVs) and NCSVs contributed to an equal proportion of patients with NDD (0.76% vs. 0.82%). The candidate genes carrying NCSVs were associated with glutamatergic synapse and chromatin remodelling. Minigene successfully validated 59 of 79 (74.68%) NCSVs that led to abnormal splicing in 40 candidate genes, and 9 of the genes (ARID1B, KAT6B, TCF4, SMARCA2, SHANK3, PDHA1, WDR45, SCN2A, SYNGAP1) harboured recurrent NCSVs with the same variant present in more than two unrelated patients with NDD. Moreover, 36 of 59 (61.02%) NCSVs are novel clinically relevant variants, including 34 unreported and 2 clinically conflicting interpretations or of uncertain significance NCSVs in the ClinVar database. INTERPRETATION This study highlights the common pathology and clinical importance of NCSVs in unsolved patients with NDD. FUNDING The present study was funded by grants from the National Natural Science Foundation of China, China Postdoctoral Science Foundation, the Hunan Youth Science and Technology Innovation Talent Project, the Provincial Natural Science Foundation of Hunan, The Scientific Research Program of FuRong laboratory, and the Natural Science Project of the University of Anhui Province.
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Affiliation(s)
- Kuokuo Li
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China; Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China
| | - Jifang Xiao
- Bioinformatics Center, National Clinical Research Centre for Geriatric Disorders, Department of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan, China; Bioinformatics Center, Furong Laboratory, Central South University, Changsha, Hunan, China
| | - Zhengbao Ling
- Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
| | - Tengfei Luo
- Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
| | - Jingyu Xiong
- Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
| | - Qian Chen
- Bioinformatics Center, National Clinical Research Centre for Geriatric Disorders, Department of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan, China; Bioinformatics Center, Furong Laboratory, Central South University, Changsha, Hunan, China
| | - Lijie Dong
- Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China; Bioinformatics Center, Furong Laboratory, Central South University, Changsha, Hunan, China
| | - Yijing Wang
- Bioinformatics Center, National Clinical Research Centre for Geriatric Disorders, Department of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan, China; Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China; Bioinformatics Center, Furong Laboratory, Central South University, Changsha, Hunan, China
| | - Xiaomeng Wang
- Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China; Bioinformatics Center, Furong Laboratory, Central South University, Changsha, Hunan, China
| | - Zhaowei Jiang
- Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
| | - Lu Xia
- Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
| | - Zhen Yu
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China; Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China
| | - Rong Hua
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China; Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China
| | - Rui Guo
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China; Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China
| | - Dongdong Tang
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China; Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China
| | - Mingrong Lv
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China; Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China
| | - Aojie Lian
- National Health Commission Key Laboratory of Birth Defect Research and Prevention, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China
| | - Bin Li
- Bioinformatics Center, National Clinical Research Centre for Geriatric Disorders, Department of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan, China; Bioinformatics Center, Furong Laboratory, Central South University, Changsha, Hunan, China
| | - GuiHu Zhao
- Bioinformatics Center, National Clinical Research Centre for Geriatric Disorders, Department of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan, China; Bioinformatics Center, Furong Laboratory, Central South University, Changsha, Hunan, China
| | - Xiaojin He
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China; Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China; Anhui Provincial Human Sperm Bank, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China.
| | - Kun Xia
- Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China.
| | - Yunxia Cao
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China; NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China; Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China.
| | - Jinchen Li
- Bioinformatics Center, National Clinical Research Centre for Geriatric Disorders, Department of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan, China; Centre for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China; Bioinformatics Center, Furong Laboratory, Central South University, Changsha, Hunan, China.
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Scheller IF, Lutz K, Mertes C, Yépez VA, Gagneur J. Improved detection of aberrant splicing with FRASER 2.0 and the intron Jaccard index. Am J Hum Genet 2023; 110:2056-2067. [PMID: 38006880 PMCID: PMC10716352 DOI: 10.1016/j.ajhg.2023.10.014] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Revised: 10/20/2023] [Accepted: 10/26/2023] [Indexed: 11/27/2023] Open
Abstract
Detection of aberrantly spliced genes is an important step in RNA-seq-based rare-disease diagnostics. We recently developed FRASER, a denoising autoencoder-based method that outperformed alternative methods of detecting aberrant splicing. However, because FRASER's three splice metrics are partially redundant and tend to be sensitive to sequencing depth, we introduce here a more robust intron-excision metric, the intron Jaccard index, that combines the alternative donor, alternative acceptor, and intron-retention signal into a single value. Moreover, we optimized model parameters and filter cutoffs by using candidate rare-splice-disrupting variants as independent evidence. On 16,213 GTEx samples, our improved algorithm, FRASER 2.0, called typically 10 times fewer splicing outliers while increasing the proportion of candidate rare-splice-disrupting variants by 10-fold and substantially decreasing the effect of sequencing depth on the number of reported outliers. To lower the multiple-testing correction burden, we introduce an option to select the genes to be tested for each sample instead of a transcriptome-wide approach. This option can be particularly useful when prior information, such as candidate variants or genes, is available. Application on 303 rare-disease samples confirmed the relative reduction in the number of outlier calls for a slight loss of sensitivity; FRASER 2.0 recovered 22 out of 26 previously identified pathogenic splicing cases with default cutoffs and 24 when multiple-testing correction was limited to OMIM genes containing rare variants. Altogether, these methodological improvements contribute to more effective RNA-seq-based rare diagnostics by drastically reducing the amount of splicing outlier calls per sample at minimal loss of sensitivity.
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Affiliation(s)
- Ines F Scheller
- School of Computation, Information and Technology, Technical University of Munich, 85748 Garching, Germany; Computational Health Center, Helmholtz Center Munich, 85764 Neuherberg, Germany
| | - Karoline Lutz
- School of Computation, Information and Technology, Technical University of Munich, 85748 Garching, Germany
| | - Christian Mertes
- School of Computation, Information and Technology, Technical University of Munich, 85748 Garching, Germany; Munich Data Science Institute, Technical University of Munich, 85748 Garching, Germany; Institute of Human Genetics, School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - Vicente A Yépez
- School of Computation, Information and Technology, Technical University of Munich, 85748 Garching, Germany.
| | - Julien Gagneur
- School of Computation, Information and Technology, Technical University of Munich, 85748 Garching, Germany; Computational Health Center, Helmholtz Center Munich, 85764 Neuherberg, Germany; Munich Data Science Institute, Technical University of Munich, 85748 Garching, Germany; Institute of Human Genetics, School of Medicine, Technical University of Munich, 81675 Munich, Germany.
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20
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Ferese R, Scala S, Suppa A, Campopiano R, Asci F, Zampogna A, Chiaravalloti MA, Griguoli A, Storto M, Pardo AD, Giardina E, Zampatti S, Fornai F, Novelli G, Fanelli M, Zecca C, Logroscino G, Centonze D, Gambardella S. Cohort analysis of novel SPAST variants in SPG4 patients and implementation of in vitro and in vivo studies to identify the pathogenic mechanism caused by splicing mutations. Front Neurol 2023; 14:1296924. [PMID: 38145127 PMCID: PMC10748595 DOI: 10.3389/fneur.2023.1296924] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Accepted: 11/14/2023] [Indexed: 12/26/2023] Open
Abstract
Introduction Pure hereditary spastic paraplegia (SPG) type 4 (SPG4) is caused by mutations of SPAST gene. This study aimed to analyze SPAST variants in SPG4 patients to highlight the occurrence of splicing mutations and combine functional studies to assess the relevance of these variants in the molecular mechanisms of the disease. Methods We performed an NGS panel in 105 patients, in silico analysis for splicing mutations, and in vitro minigene assay. Results and discussion The NGS panel was applied to screen 105 patients carrying a clinical phenotype corresponding to upper motor neuron syndrome (UMNS), selectively affecting motor control of lower limbs. Pathogenic mutations in SPAST were identified in 12 patients (11.42%), 5 missense, 3 frameshift, and 4 splicing variants. Then, we focused on the patients carrying splicing variants using a combined approach of in silico and in vitro analysis through minigene assay and RNA, if available. For two splicing variants (i.e., c.1245+1G>A and c.1414-2A>T), functional assays confirm the types of molecular alterations suggested by the in silico analysis (loss of exon 9 and exon 12). In contrast, the splicing variant c.1005-1delG differed from what was predicted (skipping exon 7), and the functional study indicates the loss of frame and formation of a premature stop codon. The present study evidenced the high splice variants in SPG4 patients and indicated the relevance of functional assays added to in silico analysis to decipher the pathogenic mechanism.
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Affiliation(s)
| | | | - Antonio Suppa
- IRCCS Neuromed, Pozzilli, Italy
- Department of Human Neurosciences, Sapienza University of Rome, Rome, Italy
| | | | | | | | | | | | | | | | - Emiliano Giardina
- Genomic Medicine Laboratory, IRCCS Fondazione Santa Lucia, Rome, Italy
| | - Stefania Zampatti
- Genomic Medicine Laboratory, IRCCS Fondazione Santa Lucia, Rome, Italy
| | - Francesco Fornai
- IRCCS Neuromed, Pozzilli, Italy
- Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy
| | - Giuseppe Novelli
- IRCCS Neuromed, Pozzilli, Italy
- Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Rome, Italy
| | - Mirco Fanelli
- Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
| | - Chiara Zecca
- Center for Neurodegenerative Diseases and the Aging Brain, Department of Clinical Research in Neurology of the University of Bari “Aldo Moro” at “Pia Fondazione Card G. Panico” Hospital Tricase, Lecce, Italy
| | - Giancarlo Logroscino
- Center for Neurodegenerative Diseases and the Aging Brain, Department of Clinical Research in Neurology of the University of Bari “Aldo Moro” at “Pia Fondazione Card G. Panico” Hospital Tricase, Lecce, Italy
| | - Diego Centonze
- IRCCS Neuromed, Pozzilli, Italy
- Department of Systems Medicine, Tor Vergata University, Rome, Italy
| | - Stefano Gambardella
- IRCCS Neuromed, Pozzilli, Italy
- Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
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21
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Suzuki Y, Nomura N, Yamada K, Yamada Y, Fukuda A, Hoshino K, Abe S, Kurosawa K, Inaba M, Mizuno S, Wakamatsu N, Hayashi S. Pathogenicity evaluation of variants of uncertain significance at exon-intron junction by splicing assay in patients with Mowat-Wilson syndrome. Eur J Med Genet 2023; 66:104882. [PMID: 37944854 DOI: 10.1016/j.ejmg.2023.104882] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Revised: 10/20/2023] [Accepted: 11/02/2023] [Indexed: 11/12/2023]
Abstract
High-throughput sequencing has identified vast numbers of variants in genetic disorders. However, the significance of variants at the exon-intron junction remains controversial. Even though most cases of Mowat-Wilson syndrome (MOWS) are caused by heterozygous loss-of-function variants in ZEB2, the pathogenicity of variants at exon-intron junction is often indeterminable. We identified four intronic variants in 5/173 patients with clinical suspicion for MOWS, and evaluated their pathogenicity by in vitro analyses. The minigene analysis showed that c.73+2T>G caused most of the transcripts skipping exon 2, while c.916+6T>G led to partial skipping of exon 7. No splicing abnormalities were detected in both c.917-21T>C and c.3067+6A>T. The minigene analysis reproduced the splicing observed in the blood cells of the patient with c.73+2T>G. The degree of the exon skipping was concordant with the severity of MOWS; while the patient with c.73+2T>G was typical MOWS, the patient with c.916+6T>G showed milder phenotype which has been seldom reported. Our results demonstrate that mRNA splicing assays using the minigenes are valuable for determining the clinical significance of intronic variants in patients with not only MOWS but also other genetic diseases with splicing aberrations and may explain atypical or milder cases, such as the current patient.
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Affiliation(s)
- Yasuyo Suzuki
- Department of Genetics, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Aichi, Japan
| | - Noriko Nomura
- Department of Genetics, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Aichi, Japan
| | - Kenichiro Yamada
- Department of Genetics, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Aichi, Japan
| | - Yasukazu Yamada
- Department of Genetics, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Aichi, Japan
| | - Ayumi Fukuda
- Department of Pediatrics, Nihon University Itabashi Hospital, Itabashi, Tokyo, Japan
| | - Kyoko Hoshino
- Segawa Memorial Neurological Clinic for Children, Chiyoda, Tokyo, Japan
| | - Shinpei Abe
- Department of Pediatrics, Juntendo University, Faculty of Medicine, Bunkyo, Tokyo, Japan
| | - Kenji Kurosawa
- Division of Medical Genetics, Kanagawa Children's Medical Center, Yokohama, Kanagawa, Japan
| | - Mie Inaba
- Department of Pediatrics, Central Hospital, Aichi Developmental Disability Center, Kasugai, Aichi, Japan
| | - Seiji Mizuno
- Department of Pediatrics, Central Hospital, Aichi Developmental Disability Center, Kasugai, Aichi, Japan
| | - Nobuaki Wakamatsu
- Department of Genetics, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Aichi, Japan; Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Takamatsu, Kagawa, Japan
| | - Shin Hayashi
- Department of Genetics, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Aichi, Japan.
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22
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Oftedal BE, Berger AH, Bruserud Ø, Goldfarb Y, Sulen A, Breivik L, Hellesen A, Ben-Dor S, Haffner-Krausz R, Knappskog PM, Johansson S, Wolff AS, Bratland E, Abramson J, Husebye ES. A partial form of AIRE deficiency underlies a mild form of autoimmune polyendocrine syndrome type 1. J Clin Invest 2023; 133:e169704. [PMID: 37909333 PMCID: PMC10617782 DOI: 10.1172/jci169704] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2023] [Accepted: 08/29/2023] [Indexed: 11/03/2023] Open
Abstract
Autoimmune polyendocrine syndrome type 1 (APS-1) is caused by mutations in the autoimmune regulator (AIRE) gene. Most patients present with severe chronic mucocutaneous candidiasis and organ-specific autoimmunity from early childhood, but the clinical picture is highly variable. AIRE is crucial for negative selection of T cells, and scrutiny of different patient mutations has previously highlighted many of its molecular mechanisms. In patients with a milder adult-onset phenotype sharing a mutation in the canonical donor splice site of intron 7 (c.879+1G>A), both the predicted altered splicing pattern with loss of exon 7 (AireEx7-/-) and normal full-length AIRE mRNA were found, indicating leaky rather than abolished mRNA splicing. Analysis of a corresponding mouse model demonstrated that the AireEx7-/- mutant had dramatically impaired transcriptional capacity of tissue-specific antigens in medullary thymic epithelial cells but still retained some ability to induce gene expression compared with the complete loss-of-function AireC313X-/- mutant. Our data illustrate an association between AIRE activity and the severity of autoimmune disease, with implications for more common autoimmune diseases associated with AIRE variants, such as primary adrenal insufficiency, pernicious anemia, type 1 diabetes, and rheumatoid arthritis.
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Affiliation(s)
- Bergithe Eikeland Oftedal
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medicine and
| | - Amund Holte Berger
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway
| | - Øyvind Bruserud
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medicine and
| | - Yael Goldfarb
- Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot, Israel
| | - Andre Sulen
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
| | - Lars Breivik
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medicine and
| | - Alexander Hellesen
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
| | - Shifra Ben-Dor
- Bioinformatics Unit, Department of Life Sciences Core Facilities and
| | | | - Per M. Knappskog
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway
| | - Stefan Johansson
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway
| | - Anette S.B. Wolff
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medicine and
| | - Eirik Bratland
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway
| | - Jakub Abramson
- Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot, Israel
| | - Eystein Sverre Husebye
- Department of Clinical Science and KG Jebsen Center for Autoimmune Diseases, University of Bergen, Norway
- Department of Medicine and
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23
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Zeibich R, Kwan P, J. O’Brien T, Perucca P, Ge Z, Anderson A. Applications for Deep Learning in Epilepsy Genetic Research. Int J Mol Sci 2023; 24:14645. [PMID: 37834093 PMCID: PMC10572791 DOI: 10.3390/ijms241914645] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 09/11/2023] [Accepted: 09/21/2023] [Indexed: 10/15/2023] Open
Abstract
Epilepsy is a group of brain disorders characterised by an enduring predisposition to generate unprovoked seizures. Fuelled by advances in sequencing technologies and computational approaches, more than 900 genes have now been implicated in epilepsy. The development and optimisation of tools and methods for analysing the vast quantity of genomic data is a rapidly evolving area of research. Deep learning (DL) is a subset of machine learning (ML) that brings opportunity for novel investigative strategies that can be harnessed to gain new insights into the genomic risk of people with epilepsy. DL is being harnessed to address limitations in accuracy of long-read sequencing technologies, which improve on short-read methods. Tools that predict the functional consequence of genetic variation can represent breaking ground in addressing critical knowledge gaps, while methods that integrate independent but complimentary data enhance the predictive power of genetic data. We provide an overview of these DL tools and discuss how they may be applied to the analysis of genetic data for epilepsy research.
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Affiliation(s)
- Robert Zeibich
- Department of Neuroscience, Central Clinical School, Monash University, Melbourne, VIC 3800, Australia; (R.Z.); (P.K.); (T.J.O.); (P.P.)
| | - Patrick Kwan
- Department of Neuroscience, Central Clinical School, Monash University, Melbourne, VIC 3800, Australia; (R.Z.); (P.K.); (T.J.O.); (P.P.)
- Department of Neurology, Alfred Health, Melbourne, VIC 3004, Australia
- Department of Neurology, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3052, Australia
- Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3052, Australia
| | - Terence J. O’Brien
- Department of Neuroscience, Central Clinical School, Monash University, Melbourne, VIC 3800, Australia; (R.Z.); (P.K.); (T.J.O.); (P.P.)
- Department of Neurology, Alfred Health, Melbourne, VIC 3004, Australia
- Department of Neurology, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3052, Australia
- Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3052, Australia
| | - Piero Perucca
- Department of Neuroscience, Central Clinical School, Monash University, Melbourne, VIC 3800, Australia; (R.Z.); (P.K.); (T.J.O.); (P.P.)
- Department of Neurology, Alfred Health, Melbourne, VIC 3004, Australia
- Department of Neurology, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3052, Australia
- Epilepsy Research Centre, Department of Medicine, Austin Health, The University of Melbourne, Melbourne, VIC 3084, Australia
- Bladin-Berkovic Comprehensive Epilepsy Program, Department of Neurology, Austin Health, The University of Melbourne, Melbourne, VIC 3084, Australia
| | - Zongyuan Ge
- Faculty of Engineering, Monash University, Melbourne, VIC 3800, Australia;
- Monash-Airdoc Research, Monash University, Melbourne, VIC 3800, Australia
| | - Alison Anderson
- Department of Neuroscience, Central Clinical School, Monash University, Melbourne, VIC 3800, Australia; (R.Z.); (P.K.); (T.J.O.); (P.P.)
- Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3052, Australia
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24
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Darbinian N, Gallia GL, Darbinyan A, Vadachkoria E, Merabova N, Moore A, Goetzl L, Amini S, Selzer ME. Effects of In Utero EtOH Exposure on 18S Ribosomal RNA Processing: Contribution to Fetal Alcohol Spectrum Disorder. Int J Mol Sci 2023; 24:13714. [PMID: 37762017 PMCID: PMC10531167 DOI: 10.3390/ijms241813714] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2023] [Revised: 08/28/2023] [Accepted: 08/29/2023] [Indexed: 09/29/2023] Open
Abstract
Fetal alcohol spectrum disorders (FASD) are leading causes of neurodevelopmental disability. The mechanisms by which alcohol (EtOH) disrupts fetal brain development are incompletely understood, as are the genetic factors that modify individual vulnerability. Because the phenotype abnormalities of FASD are so varied and widespread, we investigated whether fetal exposure to EtOH disrupts ribosome biogenesis and the processing of pre-ribosomal RNAs and ribosome assembly, by determining the effect of exposure to EtOH on the developmental expression of 18S rRNA and its cleaved forms, members of a novel class of short non-coding RNAs (srRNAs). In vitro neuronal cultures and fetal brains (11-22 weeks) were collected according to an IRB-approved protocol. Twenty EtOH-exposed brains from the first and second trimester were compared with ten unexposed controls matched for gestational age and fetal gender. Twenty fetal-brain-derived exosomes (FB-Es) were isolated from matching maternal blood. RNA was isolated using Qiagen RNA isolation kits. Fetal brain srRNA expression was quantified by ddPCR. srRNAs were expressed in the human brain and FB-Es during fetal development. EtOH exposure slightly decreased srRNA expression (1.1-fold; p = 0.03). Addition of srRNAs to in vitro neuronal cultures inhibited EtOH-induced caspase-3 activation (1.6-fold, p = 0.002) and increased cell survival (4.7%, p = 0.034). The addition of exogenous srRNAs reversed the EtOH-mediated downregulation of srRNAs (2-fold, p = 0.002). EtOH exposure suppressed expression of srRNAs in the developing brain, increased activity of caspase-3, and inhibited neuronal survival. Exogenous srRNAs reversed this effect, possibly by stabilizing endogenous srRNAs, or by increasing the association of cellular proteins with srRNAs, modifying gene transcription. Finally, the reduction in 18S rRNA levels correlated closely with the reduction in fetal eye diameter, an anatomical hallmark of FASD. The findings suggest a potential mechanism for EtOH-mediated neurotoxicity via alterations in 18S rRNA processing and the use of FB-Es for early diagnosis of FASD. Ribosome biogenesis may be a novel target to ameliorate FASD in utero or after birth. These findings are consistent with observations that gene-environment interactions contribute to FASD vulnerability.
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Affiliation(s)
- Nune Darbinian
- Center for Neural Repair and Rehabilitation Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; (E.V.); (N.M.); (A.M.)
| | - Gary L. Gallia
- Department of Neurosurgery, Johns Hopkins Hospital, Baltimore, MD 21287, USA;
| | - Armine Darbinyan
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA;
| | - Ekaterina Vadachkoria
- Center for Neural Repair and Rehabilitation Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; (E.V.); (N.M.); (A.M.)
| | - Nana Merabova
- Center for Neural Repair and Rehabilitation Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; (E.V.); (N.M.); (A.M.)
- Medical College of Wisconsin-Prevea Health, Green Bay, WI 54304, USA
| | - Amos Moore
- Center for Neural Repair and Rehabilitation Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; (E.V.); (N.M.); (A.M.)
| | - Laura Goetzl
- Department of Obstetrics & Gynecology, University of Texas, Houston, TX 77030, USA;
| | - Shohreh Amini
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, PA 19122, USA;
| | - Michael E. Selzer
- Center for Neural Repair and Rehabilitation Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; (E.V.); (N.M.); (A.M.)
- Departments of Neurology and Neural Sciences, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 19140, USA
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25
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Bouvet D, Blondel A, de Sainte Agathe JM, Leroy G, Saint-Martin C, Bellanné-Chantelot C. Evaluation in Monogenic Diabetes of the Impact of GCK, HNF1A, and HNF4A Variants on Splicing through the Combined Use of In Silico Tools and Minigene Assays. Hum Mutat 2023; 2023:6661013. [PMID: 40225161 PMCID: PMC11919142 DOI: 10.1155/2023/6661013] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2023] [Revised: 08/08/2023] [Accepted: 08/17/2023] [Indexed: 04/15/2025]
Abstract
Variants in GCK, HNF1A, and HNF4A genes are the three main causes of monogenic diabetes. Determining the molecular etiology is essential for patients with monogenic diabetes to benefit from the most appropriate treatment. The increasing number of variants of unknown significance (VUS) is a major issue in genetic diagnosis, and assessing the impact of variants on RNA splicing is challenging, particularly for genes expressed in tissues not easily accessible as in monogenic diabetes. The in vitro functional splicing assay based on a minigene construct is an appropriate approach. Here, we performed in silico analysis using SpliceAI and SPiP and prioritized 36 spliceogenic variants in GCK, HNF1A, and HNF4A. Predictions were secondarily compared with Pangolin and AbSplice-DNA bioinformatics tools which include tissue-specific annotations. We assessed the effect of selected variants on RNA splicing using minigene assays. These assays validated splicing defects for 33 out of 36 spliceogenic variants consisting of exon skipping (15%), exonic deletions (18%), intronic retentions (24%), and complex splicing patterns (42%). This provided additional evidence to reclassify 23 out of 31 (74%) VUS including missense, synonymous, and intronic noncanonical splice site variants as likely pathogenic variants. Comparison of in silico analysis with minigene results showed the robustness of bioinformatics tools to prioritize spliceogenic variants, but revealed inconsistencies in the location of cryptic splice sites underlying the importance of confirming predicted splicing alterations with functional splicing assays. Our study underlines the feasibility and the benefits of implementing minigene-splicing assays in the genetic testing of monogenic diabetes after a prior in-depth in silico analysis.
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Affiliation(s)
- Delphine Bouvet
- Department of Medical Genetics, AP-HP Pitié-Salpêtrière Hospital, Sorbonne University, Paris, France
| | - Amélie Blondel
- Department of Medical Genetics, AP-HP Pitié-Salpêtrière Hospital, Sorbonne University, Paris, France
| | | | - Gwendoline Leroy
- Department of Medical Genetics, AP-HP Pitié-Salpêtrière Hospital, Sorbonne University, Paris, France
| | - Cécile Saint-Martin
- Department of Medical Genetics, AP-HP Pitié-Salpêtrière Hospital, Sorbonne University, Paris, France
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26
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Liu X, Shi X, Xin Q, Liu Z, Pan F, Qiao D, Chen M, Zhang Y, Guo W, Li C, Zhang Y, Shao L, Zhang R. Identified eleven exon variants in PKD1 and PKD2 genes that altered RNA splicing by minigene assay. BMC Genomics 2023; 24:407. [PMID: 37468838 PMCID: PMC10354997 DOI: 10.1186/s12864-023-09444-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2023] [Accepted: 06/11/2023] [Indexed: 07/21/2023] Open
Abstract
BACKGROUND Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic multisystem disease caused primarily by mutations in the PKD1 gene or PKD2 gene. There is increasing evidence that some of these variants, which are described as missense, synonymous or nonsense mutations in the literature or databases, may be deleterious by affecting the pre-mRNA splicing process. RESULTS This study aimed to determine the effect of these PKD1 and PKD2 variants on exon splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 19 candidate single nucleotide alterations, 11 variants distributed in PKD1 (c.7866C > A, c.7960A > G, c.7979A > T, c.7987C > T, c.11248C > G, c.11251C > T, c.11257C > G, c.11257C > T, c.11346C > T, and c.11393C > G) and PKD2 (c.1480G > T) were identified to result in exon skipping. CONCLUSIONS We confirmed that 11 variants in the gene of PKD1 and PKD2 affect normal splicing by interfering the recognition of classical splicing sites or by disrupting exon splicing enhancers and generating exon splicing silencers. This is the most comprehensive study to date on pre-mRNA splicing of exonic variants in ADPKD-associated disease-causing genes in consideration of the increasing number of identified variants in PKD1 and PKD2 gene in recent years. These results emphasize the significance of assessing the effect of exon single nucleotide variants in ADPKD at the mRNA level.
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Affiliation(s)
- Xuyan Liu
- Department of Nephrology, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China
| | - Xiaomeng Shi
- Department of Nephrology, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China
| | - Qing Xin
- Department of Nephrology, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China
| | - Zhiying Liu
- Renal Division, Peking University First Hospital, Beijing, China
| | - Fengjiao Pan
- Department of Nephrology, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China
| | - Dan Qiao
- Department of Nephrology, Dalian Medical University, Dalian, China
| | - Mengke Chen
- Department of Nephrology, Shandong First Medical University, Taian, China
| | - Yiyin Zhang
- Department of Nephrology, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China
| | - Wencong Guo
- Department of Nephrology, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China
| | - Changying Li
- Department of Nephrology, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China
| | - Yan Zhang
- Department of Nephrology, Weifang Medical University, Weifang, China
| | - Leping Shao
- Department of Nephrology, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China.
| | - Ruixiao Zhang
- Department of Emergency, the Affiliated Qingdao Municipal Hospital of Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, China.
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27
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Zheng Z, Song Y, Tan X. Deciphering hERG Mutation in Long QT Syndrome Type 2 Using Antisense Oligonucleotide-Mediated Techniques: Lessons from Cystic Fibrosis. Heart Rhythm 2023:S1547-5271(23)02180-X. [PMID: 37121422 DOI: 10.1016/j.hrthm.2023.04.021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Revised: 04/18/2023] [Accepted: 04/25/2023] [Indexed: 05/02/2023]
Abstract
Long QT syndrome type 2 (LQT2) is a genetic disorder caused by mutations in the KCNH2 gene, also known as the human ether-a-go-go-related gene (hERG). Over 30% of hERG mutations result in a premature termination codon (PTC) that triggers a process called nonsense-mediated mRNA decay (NMD), where the mRNA transcript is degraded. NMD is a quality control mechanism that removes faulty mRNA to prevent the translation of truncated proteins. Recent advances in antisense oligonucleotide (ASO) technology in the field of cystic fibrosis (CF) have yielded significant progress, including the ASO-mediated comprehensive characterization of key NMD factors and exon-skipping therapy. These advances have contributed to our understanding of the role of PTC-containing mutations in disease phenotypes and have also led to the development of potentially useful therapeutic strategies. Historically, studies of CF have provided valuable insights for the research on LQT2, particularly concerning increasing the expression of hERG. In this article, we outline the current state of knowledge regarding ASO, NMD, and hERG and discuss the introduction of ASO technology in the CF to elucidate the pathogenic mechanisms through targeting NMD. We also discuss the potential clinical therapeutic benefits and limitations of ASO for the management of LQT2. By drawing on lessons learned from CF research, we explore the potential translational values of these advances into LQT2 studies.
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Affiliation(s)
- Zequn Zheng
- Department of Cardiology, Shantou University Medical College, Shantou, China; Department of Cardiology, First Affiliated Hospital of Shantou University Medical College, Shantou, China; Clinical Research Center, First Affiliated Hospital of Shantou University Medical College, Shantou, China.
| | - Yongfei Song
- Ningbo Institute for Medicine &Biomedical Engineering Combined Innovation, Ningbo, China
| | - Xuerui Tan
- Department of Cardiology, First Affiliated Hospital of Shantou University Medical College, Shantou, China; Clinical Research Center, First Affiliated Hospital of Shantou University Medical College, Shantou, China.
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28
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Levacher C, Viennot M, Drouet A, Beaussire L, Coutant S, Théry JC, Baert-Desurmont S, Laé M, Ruminy P, Houdayer C. Disequilibrium between BRCA1 and BRCA2 Circular and Messenger RNAs Plays a Role in Breast Cancer. Cancers (Basel) 2023; 15:2176. [PMID: 37046838 PMCID: PMC10093293 DOI: 10.3390/cancers15072176] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2023] [Revised: 03/30/2023] [Accepted: 03/31/2023] [Indexed: 04/14/2023] Open
Abstract
Breast cancer is a frequent disease for which the discovery of markers that enable early detection or prognostic assessment remains challenging. Circular RNAs (circRNAs) are single-stranded structures in closed loops that are produced by backsplicing. CircRNA and messenger RNA (mRNA) are generated co-transcriptionally, and backsplicing and linear splicing compete against each other. As mRNAs are key players in tumorigenesis, we hypothesize that a disruption of the balance between circRNAs and mRNAs could promote breast cancer. Hence, we developed an assay for a simultaneous study of circRNAs and mRNAs, which we have called splice and expression analyses by exon ligation and high-throughput sequencing (SEALigHTS). Following SEALigHTS validation for BRCA1 and BRCA2, our hypothesis was tested using an independent research set of 95 pairs from tumor and adjacent normal breast tissues. In this research set, ratios of BRCA1 and BRCA2 circRNAs/mRNAs were significantly lower in the tumor breast tissue compared to normal tissue (p = 1.6 × 10-9 and p = 4.4 × 10-5 for BRCA1 and BRCA2, respectively). Overall, we developed an innovative method to study linear splicing and backsplicing, described the repertoire of BRCA1 and BRCA2 circRNAs, including 15 novel ones, and showed for the first time that a disequilibrium between BRCA1 and BRCA2 circRNAs and mRNAs plays a role in breast cancer.
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Affiliation(s)
- Corentin Levacher
- Univ Rouen Normandie, INSERM U1245, FHU-G4 Génomique, 76000 Rouen, France; (C.L.)
| | - Mathieu Viennot
- Univ Rouen Normandie, INSERM U1245, Centre Henri Becquerel, 76000 Rouen, France (M.L.); (P.R.)
| | - Aurélie Drouet
- Univ Rouen Normandie, INSERM U1245, FHU-G4 Génomique, 76000 Rouen, France; (C.L.)
| | - Ludivine Beaussire
- Univ Rouen Normandie, INSERM U1245, FHU-G4 Génomique, 76000 Rouen, France; (C.L.)
- Department of Pathology, Centre Henri Becquerel, 1 Rue d’Amiens, 76038 Rouen, France
| | - Sophie Coutant
- Univ Rouen Normandie, INSERM U1245, FHU-G4 Génomique, 76000 Rouen, France; (C.L.)
| | - Jean-Christophe Théry
- Univ Rouen Normandie, INSERM U1245, FHU-G4 Génomique, 76000 Rouen, France; (C.L.)
- Department of Medical Oncology, Centre Henri Becquerel, 1 Rue d’Amiens, 76038 Rouen, France
| | - Stéphanie Baert-Desurmont
- Univ Rouen Normandie, INSERM U1245, FHU-G4 Génomique and CHU Rouen, Department of Genetics, 76000 Rouen, France
| | - Marick Laé
- Univ Rouen Normandie, INSERM U1245, Centre Henri Becquerel, 76000 Rouen, France (M.L.); (P.R.)
- Department of Pathology, Centre Henri Becquerel, 1 Rue d’Amiens, 76038 Rouen, France
| | - Philippe Ruminy
- Univ Rouen Normandie, INSERM U1245, Centre Henri Becquerel, 76000 Rouen, France (M.L.); (P.R.)
| | - Claude Houdayer
- Univ Rouen Normandie, INSERM U1245, FHU-G4 Génomique and CHU Rouen, Department of Genetics, 76000 Rouen, France
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29
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Scheller IF, Lutz K, Mertes C, Yépez VA, Gagneur J. Improved detection of aberrant splicing using the Intron Jaccard Index. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2023:2023.03.31.23287997. [PMID: 37066374 PMCID: PMC10104204 DOI: 10.1101/2023.03.31.23287997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 04/29/2023]
Abstract
Detection of aberrantly spliced genes is an important step in RNA-seq-based rare disease diagnostics. We recently developed FRASER, a denoising autoencoder-based method for aberrant splicing detection that outperformed alternative approaches. However, as FRASER's three splice metrics are partially redundant and tend to be sensitive to sequencing depth, we introduce here a more robust intron excision metric, the Intron Jaccard Index, that combines alternative donor, alternative acceptor, and intron retention signal into a single value. Moreover, we optimized model parameters and filter cutoffs using candidate rare splice-disrupting variants as independent evidence. On 16,213 GTEx samples, our improved algorithm called typically 10 times fewer splicing outliers while increasing the proportion of candidate rare splice-disrupting variants by 10 fold and substantially decreasing the effect of sequencing depth on the number of reported outliers. Application on 303 rare disease samples confirmed the reduction fold-change of the number of outlier calls for a slight loss of sensitivity (only 2 out of 22 previously identified pathogenic splicing cases not recovered). Altogether, these methodological improvements contribute to more effective RNA-seq-based rare diagnostics by a drastic reduction of the amount of splicing outlier calls per sample at minimal loss of sensitivity.
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Affiliation(s)
- Ines F. Scheller
- School of Computation, Information and Technology, Technical University of Munich, Garching, 85748, Germany
- Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, 85764, Germany
| | - Karoline Lutz
- School of Computation, Information and Technology, Technical University of Munich, Garching, 85748, Germany
| | - Christian Mertes
- School of Computation, Information and Technology, Technical University of Munich, Garching, 85748, Germany
- Munich Data Science Institute, Technical University of Munich, Garching, 85748, Germany
- Institute of Human Genetics, School of Medicine, Technical University of Munich, Munich, 81675, Germany
| | - Vicente A. Yépez
- School of Computation, Information and Technology, Technical University of Munich, Garching, 85748, Germany
| | - Julien Gagneur
- School of Computation, Information and Technology, Technical University of Munich, Garching, 85748, Germany
- Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, 85764, Germany
- Munich Data Science Institute, Technical University of Munich, Garching, 85748, Germany
- Institute of Human Genetics, School of Medicine, Technical University of Munich, Munich, 81675, Germany
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30
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Fackenthal JD. Alternative mRNA Splicing and Promising Therapies in Cancer. Biomolecules 2023; 13:biom13030561. [PMID: 36979496 PMCID: PMC10046298 DOI: 10.3390/biom13030561] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2023] [Revised: 03/09/2023] [Accepted: 03/16/2023] [Indexed: 03/30/2023] Open
Abstract
Cancer is among the leading causes of mortality worldwide. While considerable attention has been given to genetic and epigenetic sources of cancer-specific cellular activities, the role of alternative mRNA splicing has only recently received attention as a major contributor to cancer initiation and progression. The distribution of alternate mRNA splicing variants in cancer cells is different from their non-cancer counterparts, and cancer cells are more sensitive than non-cancer cells to drugs that target components of the splicing regulatory network. While many of the alternatively spliced mRNAs in cancer cells may represent "noise" from splicing dysregulation, certain recurring splicing variants have been shown to contribute to tumor progression. Some pathogenic splicing disruption events result from mutations in cis-acting splicing regulatory sequences in disease-associated genes, while others may result from shifts in balance among naturally occurring alternate splicing variants among mRNAs that participate in cell cycle progression and the regulation of apoptosis. This review provides examples of cancer-related alternate splicing events resulting from each step of mRNA processing and the promising therapies that may be used to address them.
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Affiliation(s)
- James D Fackenthal
- Department of Biological Sciences, College of Science and Health, Benedictine University, Lisle, IL 60532, USA
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31
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de Sainte Agathe JM, Filser M, Isidor B, Besnard T, Gueguen P, Perrin A, Van Goethem C, Verebi C, Masingue M, Rendu J, Cossée M, Bergougnoux A, Frobert L, Buratti J, Lejeune É, Le Guern É, Pasquier F, Clot F, Kalatzis V, Roux AF, Cogné B, Baux D. SpliceAI-visual: a free online tool to improve SpliceAI splicing variant interpretation. Hum Genomics 2023; 17:7. [PMID: 36765386 PMCID: PMC9912651 DOI: 10.1186/s40246-023-00451-1] [Citation(s) in RCA: 77] [Impact Index Per Article: 38.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2022] [Accepted: 01/18/2023] [Indexed: 02/12/2023] Open
Abstract
SpliceAI is an open-source deep learning splicing prediction algorithm that has demonstrated in the past few years its high ability to predict splicing defects caused by DNA variations. However, its outputs present several drawbacks: (1) although the numerical values are very convenient for batch filtering, their precise interpretation can be difficult, (2) the outputs are delta scores which can sometimes mask a severe consequence, and (3) complex delins are most often not handled. We present here SpliceAI-visual, a free online tool based on the SpliceAI algorithm, and show how it complements the traditional SpliceAI analysis. First, SpliceAI-visual manipulates raw scores and not delta scores, as the latter can be misleading in certain circumstances. Second, the outcome of SpliceAI-visual is user-friendly thanks to the graphical presentation. Third, SpliceAI-visual is currently one of the only SpliceAI-derived implementations able to annotate complex variants (e.g., complex delins). We report here the benefits of using SpliceAI-visual and demonstrate its relevance in the assessment/modulation of the PVS1 classification criteria. We also show how SpliceAI-visual can elucidate several complex splicing defects taken from the literature but also from unpublished cases. SpliceAI-visual is available as a Google Colab notebook and has also been fully integrated in a free online variant interpretation tool, MobiDetails ( https://mobidetails.iurc.montp.inserm.fr/MD ).
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Affiliation(s)
- Jean-Madeleine de Sainte Agathe
- Département de Génétique Médicale, Groupe Hospitalier Universitaire de la Pitié Salpêtrière, AP-HP.Sorbonne Université, Laboratoire de Médecine Génomique Sorbonne Université, Paris, France.
- Laboratoire de Biologie Médicale Multi-Sites SeqOIA (laboratoire-seqoia.fr/), Paris, France.
| | - Mathilde Filser
- Département de Génétique Médicale, Groupe Hospitalier Universitaire de la Pitié Salpêtrière, AP-HP.Sorbonne Université, Laboratoire de Médecine Génomique Sorbonne Université, Paris, France
| | - Bertrand Isidor
- Nantes Université, CHU Nantes, Service de Génétique Médicale, 44000, Nantes, France
| | - Thomas Besnard
- Nantes Université, CHU Nantes, Service de Génétique Médicale, 44000, Nantes, France
| | - Paul Gueguen
- Laboratoire de Biologie Médicale Multi-Sites SeqOIA (laboratoire-seqoia.fr/), Paris, France
- Service de Génétique, Inserm U1253, CHRU de Tours, Tours, France
| | - Aurélien Perrin
- Laboratoire de Génétique Moléculaire, CHU de Montpellier, Université de Montpellier, Montpellier, France
| | - Charles Van Goethem
- Laboratoire de Génétique Moléculaire, CHU de Montpellier, Université de Montpellier, Montpellier, France
| | - Camille Verebi
- Service de Médecine Génomique, Maladies de Système et d'Organe, Fédération de Génétique et de Médecine Génomique, DMU BioPhyGen, APHP Centre-Université Paris Cité, Hôpital Cochin, Paris, France
| | - Marion Masingue
- Centre de référence des maladies neuromusculaires Nord/Est/Ile de France, Hôpital Pitié-Salpêtrière, APHP, Paris, France
| | - John Rendu
- Inserm, U1216, CHU Grenoble Alpes, Grenoble Institut Neurosciences, Université Grenoble Alpes, Grenoble, France
| | - Mireille Cossée
- Laboratoire de Génétique Moléculaire, CHU de Montpellier, Université de Montpellier, Montpellier, France
- PhyMedExp, INSERM, CNRS, Université de Montpellier, Montpellier, France
| | - Anne Bergougnoux
- Laboratoire de Génétique Moléculaire, CHU de Montpellier, Université de Montpellier, Montpellier, France
- PhyMedExp, INSERM, CNRS, Université de Montpellier, Montpellier, France
| | - Laurent Frobert
- Laboratoire de Biologie Médicale Multi-Sites SeqOIA (laboratoire-seqoia.fr/), Paris, France
| | - Julien Buratti
- Département de Génétique Médicale, Groupe Hospitalier Universitaire de la Pitié Salpêtrière, AP-HP.Sorbonne Université, Laboratoire de Médecine Génomique Sorbonne Université, Paris, France
| | - Élodie Lejeune
- Département de Génétique Médicale, Groupe Hospitalier Universitaire de la Pitié Salpêtrière, AP-HP.Sorbonne Université, Laboratoire de Médecine Génomique Sorbonne Université, Paris, France
| | - Éric Le Guern
- Département de Génétique Médicale, Groupe Hospitalier Universitaire de la Pitié Salpêtrière, AP-HP.Sorbonne Université, Laboratoire de Médecine Génomique Sorbonne Université, Paris, France
- Laboratoire de Biologie Médicale Multi-Sites SeqOIA (laboratoire-seqoia.fr/), Paris, France
| | - Florence Pasquier
- Centre mémoire, Inserm U1172 DistALZ, Licend, Univ Lille, CHU Lille, 59000, Lille, France
| | - Fabienne Clot
- Département de Génétique Médicale, Groupe Hospitalier Universitaire de la Pitié Salpêtrière, AP-HP.Sorbonne Université, Laboratoire de Médecine Génomique Sorbonne Université, Paris, France
| | | | - Anne-Françoise Roux
- Laboratoire de Génétique Moléculaire, CHU de Montpellier, Université de Montpellier, Montpellier, France
- INM, Univ Montpellier, INSERM, CHU Montpellier, Montpellier, France
| | - Benjamin Cogné
- Laboratoire de Biologie Médicale Multi-Sites SeqOIA (laboratoire-seqoia.fr/), Paris, France
- Nantes Université, CHU Nantes, Service de Génétique Médicale, 44000, Nantes, France
| | - David Baux
- Laboratoire de Génétique Moléculaire, CHU de Montpellier, Université de Montpellier, Montpellier, France
- INM, Univ Montpellier, INSERM, CHU Montpellier, Montpellier, France
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32
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Dawes R, Bournazos AM, Bryen SJ, Bommireddipalli S, Marchant RG, Joshi H, Cooper ST. SpliceVault predicts the precise nature of variant-associated mis-splicing. Nat Genet 2023; 55:324-332. [PMID: 36747048 PMCID: PMC9925382 DOI: 10.1038/s41588-022-01293-8] [Citation(s) in RCA: 41] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Accepted: 12/15/2022] [Indexed: 02/08/2023]
Abstract
Even for essential splice-site variants that are almost guaranteed to alter mRNA splicing, no current method can reliably predict whether exon-skipping, cryptic activation or multiple events will result, greatly complicating clinical interpretation of pathogenicity. Strikingly, ranking the four most common unannotated splicing events across 335,663 reference RNA-sequencing (RNA-seq) samples (300K-RNA Top-4) predicts the nature of variant-associated mis-splicing with 92% sensitivity. The 300K-RNA Top-4 events correctly identify 96% of exon-skipping events and 86% of cryptic splice sites for 140 clinical cases subject to RNA testing, showing higher sensitivity and positive predictive value than SpliceAI. Notably, RNA re-analyses showed we had missed 300K-RNA Top-4 events for several clinical cases tested before the development of this empirical predictive method. Simply, mis-splicing events that happen around a splice site in RNA-seq data are those most likely to be activated by a splice-site variant. The SpliceVault web portal allows users easy access to 300K-RNA for informed splice-site variant interpretation and classification.
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Affiliation(s)
- Ruebena Dawes
- Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Sydney, New South Wales, Australia
- Discipline of Child and Adolescent Health, Faculty of Health and Medicine, University of Sydney, Sydney, New South Wales, Australia
- The Children's Medical Research Institute, Sydney, New South Wales, Australia
| | - Adam M Bournazos
- Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Sydney, New South Wales, Australia
- Discipline of Child and Adolescent Health, Faculty of Health and Medicine, University of Sydney, Sydney, New South Wales, Australia
- The Children's Medical Research Institute, Sydney, New South Wales, Australia
| | - Samantha J Bryen
- Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Sydney, New South Wales, Australia
- Discipline of Child and Adolescent Health, Faculty of Health and Medicine, University of Sydney, Sydney, New South Wales, Australia
- The Children's Medical Research Institute, Sydney, New South Wales, Australia
| | - Shobhana Bommireddipalli
- Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Sydney, New South Wales, Australia
- The Children's Medical Research Institute, Sydney, New South Wales, Australia
| | - Rhett G Marchant
- Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Sydney, New South Wales, Australia
- Discipline of Child and Adolescent Health, Faculty of Health and Medicine, University of Sydney, Sydney, New South Wales, Australia
- The Children's Medical Research Institute, Sydney, New South Wales, Australia
| | - Himanshu Joshi
- Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Sydney, New South Wales, Australia
- The Children's Medical Research Institute, Sydney, New South Wales, Australia
| | - Sandra T Cooper
- Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Sydney, New South Wales, Australia.
- Discipline of Child and Adolescent Health, Faculty of Health and Medicine, University of Sydney, Sydney, New South Wales, Australia.
- The Children's Medical Research Institute, Sydney, New South Wales, Australia.
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33
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Lightfoot HL, Smith GF. Targeting RNA with small molecules-A safety perspective. Br J Pharmacol 2023. [PMID: 36631428 DOI: 10.1111/bph.16027] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Revised: 06/30/2022] [Accepted: 12/20/2022] [Indexed: 01/13/2023] Open
Abstract
RNA is a major player in cellular function, and consequently can drive a number of disease pathologies. Over the past several years, small molecule-RNA targeting (smRNA targeting) has developed into a promising drug discovery approach. Numerous techniques, tools, and assays have been developed to support this field, and significant investments have been made by pharmaceutical and biotechnology companies. To date, the focus has been on identifying disease validated primary targets for smRNA drug development, yet RNA as a secondary (off) target for all small molecule drug programs largely has been unexplored. In this perspective, we discuss structure, target, and mechanism-driven safety aspects of smRNAs and highlight how these parameters can be evaluated in drug discovery programs to produce potentially safer drugs.
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Affiliation(s)
- Helen L Lightfoot
- Safety and Mechanistic Pharmacology, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, UK
| | - Graham F Smith
- Data Science and AI, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, UK
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34
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Leman R, Parfait B, Vidaud D, Girodon E, Pacot L, Le Gac G, Ka C, Ferec C, Fichou Y, Quesnelle C, Aucouturier C, Muller E, Vaur D, Castera L, Boulouard F, Ricou A, Tubeuf H, Soukarieh O, Gaildrat P, Riant F, Guillaud‐Bataille M, Caputo SM, Caux‐Moncoutier V, Boutry‐Kryza N, Bonnet‐Dorion F, Schultz I, Rossing M, Quenez O, Goldenberg L, Harter V, Parsons MT, Spurdle AB, Frébourg T, Martins A, Houdayer C, Krieger S. SPiP: Splicing Prediction Pipeline, a machine learning tool for massive detection of exonic and intronic variant effects on mRNA splicing. Hum Mutat 2022; 43:2308-2323. [PMID: 36273432 PMCID: PMC10946553 DOI: 10.1002/humu.24491] [Citation(s) in RCA: 34] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2022] [Revised: 10/06/2022] [Accepted: 10/18/2022] [Indexed: 01/25/2023]
Abstract
Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5'/3' splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high-throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/.
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Affiliation(s)
- Raphaël Leman
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
- UNICAENNormandie UniversitéCaenFrance
| | - Béatrice Parfait
- Service de Génétique et Biologie Moléculaires, APHP, HUPCHôpital CochinParisFrance
| | - Dominique Vidaud
- Service de Génétique et Biologie Moléculaires, APHP, HUPCHôpital CochinParisFrance
| | - Emmanuelle Girodon
- Service de Génétique et Biologie Moléculaires, APHP, HUPCHôpital CochinParisFrance
| | - Laurence Pacot
- Service de Génétique et Biologie Moléculaires, APHP, HUPCHôpital CochinParisFrance
| | - Gérald Le Gac
- Inserm UMR1078, Genetics, Functional Genomics and BiotechnologyUniversité de Bretagne OccidentaleBrestFrance
| | - Chandran Ka
- Inserm UMR1078, Genetics, Functional Genomics and BiotechnologyUniversité de Bretagne OccidentaleBrestFrance
| | - Claude Ferec
- Inserm UMR1078, Genetics, Functional Genomics and BiotechnologyUniversité de Bretagne OccidentaleBrestFrance
| | - Yann Fichou
- Inserm UMR1078, Genetics, Functional Genomics and BiotechnologyUniversité de Bretagne OccidentaleBrestFrance
| | - Céline Quesnelle
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
| | - Camille Aucouturier
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | - Etienne Muller
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
| | - Dominique Vaur
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | - Laurent Castera
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | - Flavie Boulouard
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | - Agathe Ricou
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | - Hélène Tubeuf
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
- Integrative BiosoftwareRouenFrance
| | - Omar Soukarieh
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | | | - Florence Riant
- Laboratoire de Génétique, AP‐HPGH Saint‐Louis‐Lariboisière‐Fernand WidalParisFrance
| | | | - Sandrine M. Caputo
- Department of Genetics, Institut CurieParis Sciences Lettres Research UniversityParisFrance
| | | | - Nadia Boutry‐Kryza
- Unité Mixte de Génétique Constitutionnelle des Cancers FréquentsHospices Civils de LyonLyonFrance
| | - Françoise Bonnet‐Dorion
- Departement de Biopathologie Unité de Génétique ConstitutionnelleInstitut Bergonie—INSERM U1218BordeauxFrance
| | - Ines Schultz
- Laboratoire d'OncogénétiqueCentre Paul StraussStrasbourgFrance
| | - Maria Rossing
- Centre for Genomic Medicine, RigshospitaletUniversity of CopenhagenCopenhagenDenmark
| | - Olivier Quenez
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | - Louis Goldenberg
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | - Valentin Harter
- Department of BiostatisticsBaclesse Unicancer CenterCaenFrance
| | - Michael T. Parsons
- Department of Genetics and Computational BiologyQIMR Berghofer Medical Research InstituteHerstonQueenslandAustralia
| | - Amanda B. Spurdle
- Department of Genetics and Computational BiologyQIMR Berghofer Medical Research InstituteHerstonQueenslandAustralia
| | - Thierry Frébourg
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
- Department of geneticsRouen University HospitalRouenFrance
| | - Alexandra Martins
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
| | - Claude Houdayer
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
- Department of geneticsRouen University HospitalRouenFrance
| | - Sophie Krieger
- Laboratoire de Biologie et Génétique du CancerCentre François BaclesseCaenFrance
- Inserm U1245, UNIROUEN, FHU‐G4 génomiqueNormandie UniversitéRouenFrance
- UNICAENNormandie UniversitéCaenFrance
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Identification of Novel mRNA Isoforms Associated with Acute Heat Stress Response Using RNA Sequencing Data in Sprague Dawley Rats. BIOLOGY 2022; 11:biology11121740. [PMID: 36552250 PMCID: PMC9774719 DOI: 10.3390/biology11121740] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 11/17/2022] [Accepted: 11/21/2022] [Indexed: 12/02/2022]
Abstract
The molecular mechanisms underlying heat stress tolerance in animals to high temperatures remain unclear. This study identified the differentially expressed mRNA isoforms which narrowed down the most reliable DEG markers and molecular pathways that underlie the mechanisms of thermoregulation. This experiment was performed on Sprague Dawley rats housed at 22 °C (control group; CT), and three acute heat-stressed groups housed at 42 °C for 30 min (H30), 60 min (H60), and 120 min (H120). Earlier, we demonstrated that acute heat stress increased the rectal temperature of rats, caused abnormal changes in the blood biochemical parameters, as well as induced dramatic changes in the expression levels of genes through epigenetics and post-transcriptional regulation. Transcriptomic analysis using RNA-Sequencing (RNA-Seq) data obtained previously from blood (CT and H120), liver (CT, H30, H60, and H120), and adrenal glands (CT, H30, H60, and H120) was performed. The differentially expressed mRNA isoforms (DEIs) were identified and annotated by the CLC Genomics Workbench. Biological process and metabolic pathway analyses were performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A total of 225, 5764, and 4988 DEIs in the blood, liver, and adrenal glands were observed. Furthermore, the number of novel differentially expressed transcript lengths with annotated genes and novel differentially expressed transcript with non-annotated genes were 136 and 8 in blood, 3549 and 120 in the liver, as well as 3078 and 220 in adrenal glands, respectively. About 35 genes were involved in the heat stress response, out of which, Dnaja1, LOC680121, Chordc1, AABR07011951.1, Hsp90aa1, Hspa1b, Cdkn1a, Hmox1, Bag3, and Dnaja4 were commonly identified in the liver and adrenal glands, suggesting that these genes may regulate heat stress response through interactions between the liver and adrenal glands. In conclusion, this study would enhance our understanding of the complex underlying mechanisms of acute heat stress, and the identified mRNA isoforms and genes can be used as potential candidates for thermotolerance selection in mammals.
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Zhou YQ, Wang XQ, Jiang J, Huang SL, Dai ZJ, Kong QQ. Novel hydroxymethylbilane synthase gene mutation identified and confirmed in a woman with acute intermittent porphyria: A case report. World J Clin Cases 2022; 10:12319-12327. [PMID: 36483813 PMCID: PMC9724524 DOI: 10.12998/wjcc.v10.i33.12319] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/11/2022] [Revised: 10/10/2022] [Accepted: 11/02/2022] [Indexed: 11/23/2022] Open
Abstract
BACKGROUND Acute intermittent porphyria (AIP) is a rare autosomal dominant porphyrin metabolic disease caused by a mutation in the hydroxymethylbilane synthase(HMBS) gene. This study aimed to explore the clinical manifestations of a patient with AIP, to identify a novel HMBS gene mutation in the proband and some of her family members, and to confirm the pathogenicity of the variant.
CASE SUMMARY A 22-year-old Chinese woman developed severe abdominal pain, lumbago, sinus tachycardia, epileptic seizure, hypertension, and weakness in lower limbs in March, 2018. Biochemical examinations indicated hypohepatia and hyponatremia. Her last menstrual period was 45 d prior to admission, and she was unaware of the pregnancy, which was confirmed by a pregnancy test after admission. Sunlight exposure of her urine sample for 1 h turned it from yellow to wine red. Urinary porphyrin test result was positive. Based on these clinical manifestations, AIP was diagnosed. After increasing her daily glucose intake (250–300 g/d), abdominal pain was partially relieved. Three days after hospitalization, spontaneous vaginal bleeding occurred, which was confirmed as spontaneous abortion; thereafter, her clinical symptoms completely resolved. Genetic testing revealed a novel heterozygous splicing variant of the HMBS gene in exon 10 (c.648_651+1delCCAGG) in the proband and four other family members. The pathogenicity of the variant was verified through bioinformatic methods and a minigene assay.
CONCLUSION We identified a novel HMBS gene mutation in a Chinese patient with AIP and confirmed its pathogenicity.
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Affiliation(s)
- Yu-Qing Zhou
- Department of Endocrinology, Dongguan Hospital of Traditional Chinese Medicine, Dongguan 523003, Guangdong Province, China
| | - Xiao-Qing Wang
- Department of Endocrinology, Dongguan Hospital of Traditional Chinese Medicine, Dongguan 523003, Guangdong Province, China
| | - Jun Jiang
- Department of Science and Technology ServicesChina Beijing Macro and Micro Test Biotech Co. Ltd, Beijing 100318, China
| | - Shu-Ling Huang
- Department of Endocrinology, Dongguan Hospital of Traditional Chinese Medicine, Dongguan 523003, Guangdong Province, China
| | - Zhuo-Jin Dai
- The First Clinical Medical College, Guangdong Medical University, Zhanjiang 523003, Guangdong Province, China
| | - Qiao-Qiong Kong
- Department of Medicine, Wanjiang People's Hospital of Dongguan, Dongguan 523003, Guangdong Province, China
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Cormier MJ, Pedersen BS, Bayrak-Toydemir P, Quinlan AR. Combining genetic constraint with predictions of alternative splicing to prioritize deleterious splicing in rare disease studies. BMC Bioinformatics 2022; 23:482. [PMID: 36376793 PMCID: PMC9664736 DOI: 10.1186/s12859-022-05041-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Accepted: 11/07/2022] [Indexed: 11/16/2022] Open
Abstract
BACKGROUND Despite numerous molecular and computational advances, roughly half of patients with a rare disease remain undiagnosed after exome or genome sequencing. A particularly challenging barrier to diagnosis is identifying variants that cause deleterious alternative splicing at intronic or exonic loci outside of canonical donor or acceptor splice sites. RESULTS Several existing tools predict the likelihood that a genetic variant causes alternative splicing. We sought to extend such methods by developing a new metric that aids in discerning whether a genetic variant leads to deleterious alternative splicing. Our metric combines genetic variation in the Genome Aggregate Database with alternative splicing predictions from SpliceAI to compare observed and expected levels of splice-altering genetic variation. We infer genic regions with significantly less splice-altering variation than expected to be constrained. The resulting model of regional splicing constraint captures differential splicing constraint across gene and exon categories, and the most constrained genic regions are enriched for pathogenic splice-altering variants. Building from this model, we developed ConSpliceML. This ensemble machine learning approach combines regional splicing constraint with multiple per-nucleotide alternative splicing scores to guide the prediction of deleterious splicing variants in protein-coding genes. ConSpliceML more accurately distinguishes deleterious and benign splicing variants than state-of-the-art splicing prediction methods, especially in "cryptic" splicing regions beyond canonical donor or acceptor splice sites. CONCLUSION Integrating a model of genetic constraint with annotations from existing alternative splicing tools allows ConSpliceML to prioritize potentially deleterious splice-altering variants in studies of rare human diseases.
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Affiliation(s)
- Michael J Cormier
- Department of Human Genetics, University of Utah, Salt Lake City, UT, USA
- Utah Center for Genetic Discovery, University of Utah, Salt Lake City, UT, USA
| | - Brent S Pedersen
- Department of Human Genetics, University of Utah, Salt Lake City, UT, USA
- Utah Center for Genetic Discovery, University of Utah, Salt Lake City, UT, USA
| | | | - Aaron R Quinlan
- Department of Human Genetics, University of Utah, Salt Lake City, UT, USA.
- Utah Center for Genetic Discovery, University of Utah, Salt Lake City, UT, USA.
- Department of Biomedical Informatics, University of Utah, Salt Lake City, UT, USA.
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Caceres M, Mumey B, Husic E, Rizzi R, Cairo M, Sahlin K, Tomescu AI. Safety in Multi-Assembly via Paths Appearing in All Path Covers of a DAG. IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS 2022; 19:3673-3684. [PMID: 34847041 DOI: 10.1109/tcbb.2021.3131203] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
A multi-assembly problem asks to reconstruct multiple genomic sequences from mixed reads sequenced from all of them. Standard formulations of such problems model a solution as a path cover in a directed acyclic graph, namely a set of paths that together cover all vertices of the graph. Since multi-assembly problems admit multiple solutions in practice, we consider an approach commonly used in standard genome assembly: output only partial solutions (contigs, or safe paths), that appear in all path cover solutions. We study constrained path covers, a restriction on the path cover solution that incorporate practical constraints arising in multi-assembly problems. We give efficient algorithms finding all maximal safe paths for constrained path covers. We compute the safe paths of splicing graphs constructed from transcript annotations of different species. Our algorithms run in less than 15 seconds per species and report RNA contigs that are over 99% precise and are up to 8 times longer than unitigs. Moreover, RNA contigs cover over 70% of the transcripts and their coding sequences in most cases. With their increased length to unitigs, high precision, and fast construction time, maximal safe paths can provide a better base set of sequences for transcript assembly programs.
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Skryabin NA, Zhigalina DI, Stepanov VA. The Role of Splicing in the Pathogenesis of Monogenic Diseases. RUSS J GENET+ 2022. [DOI: 10.1134/s1022795422100088] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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40
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Valenzuela-Palomo A, Sanoguera-Miralles L, Bueno-Martínez E, Esteban-Sánchez A, Llinares-Burguet I, García-Álvarez A, Pérez-Segura P, Gómez-Barrero S, de la Hoya M, Velasco-Sampedro EA. Splicing Analysis of 16 PALB2 ClinVar Variants by Minigene Assays: Identification of Six Likely Pathogenic Variants. Cancers (Basel) 2022; 14:cancers14184541. [PMID: 36139699 PMCID: PMC9496955 DOI: 10.3390/cancers14184541] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 09/12/2022] [Accepted: 09/15/2022] [Indexed: 11/29/2022] Open
Abstract
PALB2 loss-of-function variants are associated with significant increased risk of breast cancer as well as other types of tumors. Likewise, splicing disruptions are a common mechanism of disease susceptibility. Indeed, we previously showed, by minigene assays, that 35 out of 42 PALB2 variants impaired splicing. Taking advantage of one of these constructs (mgPALB2_ex1-3), we proceeded to analyze other variants at exons 1 to 3 reported at the ClinVar database. Thirty-one variants were bioinformatically analyzed with MaxEntScan and SpliceAI. Then, 16 variants were selected for subsequent RNA assays. We identified a total of 12 spliceogenic variants, 11 of which did not produce any trace of the expected minigene full-length transcript. Interestingly, variant c.49-1G > A mimicked previous outcomes in patient RNA (transcript ∆(E2p6)), supporting the reproducibility of the minigene approach. A total of eight variant-induced transcripts were characterized, three of which (∆(E1q17), ∆(E3p11), and ∆(E3)) were predicted to introduce a premature termination codon and to undergo nonsense-mediated decay, and five (▼(E1q9), ∆(E2p6), ∆(E2), ▼(E3q48)-a, and ▼(E3q48)-b) maintained the reading frame. According to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, which integrates mgPALB2 data, six PALB2 variants were classified as pathogenic/likely pathogenic, five as VUS, and five as likely benign. Furthermore, five ±1,2 variants were catalogued as VUS because they produced significant proportions of in-frame transcripts of unknown impact on protein function.
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Affiliation(s)
- Alberto Valenzuela-Palomo
- Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
| | - Lara Sanoguera-Miralles
- Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
| | - Elena Bueno-Martínez
- Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
| | - Ada Esteban-Sánchez
- Molecular Oncology Laboratory, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), 28040 Madrid, Spain
| | - Inés Llinares-Burguet
- Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
| | - Alicia García-Álvarez
- Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
| | - Pedro Pérez-Segura
- Molecular Oncology Laboratory, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), 28040 Madrid, Spain
| | - Susana Gómez-Barrero
- Facultad de Ciencias de la Salud, Universidad Alfonso X “El Sabio”, Avda. de la Universidad 1, Villanueva de la Cañada, 28691 Madrid, Spain
| | - Miguel de la Hoya
- Molecular Oncology Laboratory, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), 28040 Madrid, Spain
| | - Eladio A. Velasco-Sampedro
- Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), 47003 Valladolid, Spain
- Correspondence:
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Hatch ST, Smargon AA, Yeo GW. Engineered U1 snRNAs to modulate alternatively spliced exons. Methods 2022; 205:140-148. [PMID: 35764245 PMCID: PMC11185844 DOI: 10.1016/j.ymeth.2022.06.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2022] [Revised: 05/30/2022] [Accepted: 06/23/2022] [Indexed: 10/17/2022] Open
Abstract
Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool. With the goal of providing a user-friendly protocol, we compile and demonstrate a methodological toolkit for sequence-specific targeted perturbation of alternatively spliced pre-mRNA with engineered U1 snRNAs. We observe robust modulation of endogenous pre-mRNA transcripts targeted in two contrasting splicing contexts, SMN2 exon 7 and FAS exon 6, exhibiting the utility and applicability of engineered U1 snRNA to both inclusion and exclusion of targeted exons. We anticipate that these demonstrations will contribute to the usability of U1 snRNA in investigating splicing modulation in eukaryotic cells, increasing accessibility to the broader research community.
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Affiliation(s)
- Samuel T Hatch
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA; Stem Cell Program, University of California San Diego, Sanford Consortium for Regenerative Medicine, La Jolla, USA; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA; Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA, USA
| | - Aaron A Smargon
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA; Stem Cell Program, University of California San Diego, Sanford Consortium for Regenerative Medicine, La Jolla, USA; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA
| | - Gene W Yeo
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA; Stem Cell Program, University of California San Diego, Sanford Consortium for Regenerative Medicine, La Jolla, USA; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA; Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA, USA.
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Poly(A) capture full length cDNA sequencing improves the accuracy and detection ability of transcript quantification and alternative splicing events. Sci Rep 2022; 12:10599. [PMID: 35732903 PMCID: PMC9217819 DOI: 10.1038/s41598-022-14902-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Accepted: 06/14/2022] [Indexed: 11/08/2022] Open
Abstract
The full-length double-strand cDNA sequencing, one of the RNA-Seq methods, is a powerful method used to investigate the transcriptome status of a gene of interest, such as its transcription level and alternative splicing variants. Furthermore, full-length double-strand cDNA sequencing has the advantage that it can create a library from a small amount of sample and the library can be applied to long-read sequencers in addition to short-read sequencers. Nevertheless, one of our previous studies indicated that the full-length double-strand cDNA sequencing yields non-specific genomic DNA amplification, affecting transcriptome analysis, such as transcript quantification and alternative splicing analysis. In this study, it was confirmed that it is possible to produce the RNA-Seq library from only genomic DNA and that the full-length double-strand cDNA sequencing of genomic DNA yielded non-specific genomic DNA amplification. To avoid non-specific genomic DNA amplification, two methods were examined, which are the DNase I-treated full-length double-strand cDNA sequencing and poly(A) capture full-length double-strand cDNA sequencing. Contrary to expectations, the non-specific genomic DNA amplification was increased and the number of the detected expressing genes was reduced in DNase I-treated full-length double-strand cDNA sequencing. On the other hand, in the poly(A) capture full-length double-strand cDNA sequencing, the non-specific genomic DNA amplification was significantly reduced, accordingly the accuracy and the number of detected expressing genes and splicing events were increased. The expression pattern and percentage spliced in index of splicing events were highly correlated. Our results indicate that the poly(A) capture full-length double-strand cDNA sequencing improves transcript quantification accuracy and the detection ability of alternative splicing events. It is also expected to contribute to the determination of the significance of DNA variants to splicing events.
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CI-SpliceAI—Improving machine learning predictions of disease causing splicing variants using curated alternative splice sites. PLoS One 2022; 17:e0269159. [PMID: 35657932 PMCID: PMC9165884 DOI: 10.1371/journal.pone.0269159] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 05/16/2022] [Indexed: 11/23/2022] Open
Abstract
Background It is estimated that up to 50% of all disease causing variants disrupt splicing. Due to its complexity, our ability to predict which variants disrupt splicing is limited, meaning missed diagnoses for patients. The emergence of machine learning for targeted medicine holds great potential to improve prediction of splice disrupting variants. The recently published SpliceAI algorithm utilises deep neural networks and has been reported to have a greater accuracy than other commonly used methods. Methods and findings The original SpliceAI was trained on splice sites included in primary isoforms combined with novel junctions observed in GTEx data, which might introduce noise and de-correlate the machine learning input with its output. Limiting the data to only validated and manual annotated primary and alternatively spliced GENCODE sites in training may improve predictive abilities. All of these gene isoforms were collapsed (aggregated into one pseudo-isoform) and the SpliceAI architecture was retrained (CI-SpliceAI). Predictive performance on a newly curated dataset of 1,316 functionally validated variants from the literature was compared with the original SpliceAI, alongside MMSplice, MaxEntScan, and SQUIRLS. Both SpliceAI algorithms outperformed the other methods, with the original SpliceAI achieving an accuracy of ∼91%, and CI-SpliceAI showing an improvement at ∼92% overall. Predictive accuracy increased in the majority of curated variants. Conclusions We show that including only manually annotated alternatively spliced sites in training data improves prediction of clinically relevant variants, and highlight avenues for further performance improvements.
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Gu H, Hong J, Lee W, Kim SB, Chun S, Min WK. RNA Sequencing for Elucidating an Intronic Variant of Uncertain Significance ( SDHD c.314+3A>T) in Splicing Site Consensus Sequences. Ann Lab Med 2022; 42:376-379. [PMID: 34907111 PMCID: PMC8677484 DOI: 10.3343/alm.2022.42.3.376] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2021] [Revised: 09/02/2021] [Accepted: 11/29/2021] [Indexed: 11/19/2022] Open
Affiliation(s)
- Hyunjung Gu
- Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Jinyoung Hong
- Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Woochang Lee
- Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Sung-Bae Kim
- Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Sail Chun
- Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Won-Ki Min
- Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
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Empirical prediction of variant-activated cryptic splice donors using population-based RNA-Seq data. Nat Commun 2022; 13:1655. [PMID: 35351883 PMCID: PMC8964760 DOI: 10.1038/s41467-022-29271-y] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2021] [Accepted: 03/01/2022] [Indexed: 11/24/2022] Open
Abstract
Predicting which cryptic-donors may be activated by a splicing variant in patient DNA is notoriously difficult. Through analysis of 5145 cryptic-donors (versus 86,963 decoy-donors not used; any GT or GC), we define an empirical method predicting cryptic-donor activation with 87% sensitivity and 95% specificity. Strength (according to four algorithms) and proximity to the annotated-donor appear important determinants of cryptic-donor activation. However, other factors such as splicing regulatory elements, which are difficult to identify, play an important role and are likely responsible for current prediction inaccuracies. We find that the most frequently recurring natural mis-splicing events at each exon-intron junction, summarised over 40,233 RNA-sequencing samples (40K-RNA), predict with accuracy which cryptic-donor will be activated in rare disease. 40K-RNA provides an accurate, evidence-based method to predict variant-activated cryptic-donors in genetic disorders, assisting pathology consideration of possible consequences of a variant for the encoded protein and RNA diagnostic testing strategies. Genetic variants affecting the consensus splicing motifs can alter binding of spliceosomal components and induce mis-splicing. Here, the authors develop a method, showing that ranking the most common recurring mis-splicing events in public RNA-Seq data can predict the activation of cryptic-donors.
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Wu T, Deme L, Zhang Z, Huang X, Xu S, Yang G. Decay of TRPV3 as the genomic trace of epidermal structure changes in the land-to-sea transition of mammals. Ecol Evol 2022; 12:e8731. [PMID: 35342611 PMCID: PMC8931706 DOI: 10.1002/ece3.8731] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2021] [Revised: 02/22/2022] [Accepted: 02/24/2022] [Indexed: 11/09/2022] Open
Abstract
The epidermis plays an indispensable barrier function in animals. Some species have evolved unique epidermal structures to adapt to different environments. Aquatic and semi-aquatic mammals (cetaceans, manatees, and hippopotamus) are good models to study the evolution of epidermal structures because of their exceptionally thickened stratum spinosum, the lack of stratum granulosum, and the parakeratotic stratum corneum. This study aimed to analyze an upstream regulatory gene transient receptor potential cation channel, subfamily V, member 3 (TRPV3) of epidermal differentiation so as to explore the association between TRPV3 evolution and epidermal changes in mammals. Inactivating mutations were detected in almost all the aquatic cetaceans and several terrestrial mammals. Relaxed selective pressure was examined in the cetacean lineages with inactivated TRPV3, which might contribute to its exceptionally thickened stratum spinosum as the significant thickening of stratum spinosum in TRPV3 knock-out mouse. However, functional TRPV3 may exist in several terrestrial mammals due to their strong purifying selection, although they have "inactivating mutations." Further, for intact sequences, relaxed selective constraints on the TRPV3 gene were also detected in aquatic cetaceans, manatees, and semi-aquatic hippopotamus. However, they had intact TRPV3, suggesting that the accumulation of inactivating mutations might have lagged behind the relaxed selective pressure. The results of this study revealed the decay of TRPV3 being the genomic trace of epidermal development in aquatic and semi-aquatic mammals. They provided insights into convergently evolutionary changes of epidermal structures during the transition from the terrestrial to the aquatic environment.
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Affiliation(s)
- Tianzhen Wu
- College of Life SciencesJiangsu Key Laboratory for Biodiversity and BiotechnologyNanjing Normal UniversityNanjingChina
| | - Luoying Deme
- College of Life SciencesJiangsu Key Laboratory for Biodiversity and BiotechnologyNanjing Normal UniversityNanjingChina
| | - Zhenhua Zhang
- College of Life SciencesJiangsu Key Laboratory for Biodiversity and BiotechnologyNanjing Normal UniversityNanjingChina
| | - Xin Huang
- College of Life SciencesJiangsu Key Laboratory for Biodiversity and BiotechnologyNanjing Normal UniversityNanjingChina
| | - Shixia Xu
- College of Life SciencesJiangsu Key Laboratory for Biodiversity and BiotechnologyNanjing Normal UniversityNanjingChina
| | - Guang Yang
- College of Life SciencesJiangsu Key Laboratory for Biodiversity and BiotechnologyNanjing Normal UniversityNanjingChina
- Southern Marine Science and Engineering Guangdong LaboratoryGuangzhouChina
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47
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He WB, Xiao WJ, Dai CL, Wang YR, Li XR, Gong F, Meng LL, Tan C, Zeng SC, Lu GX, Lin G, Tan YQ, Hu H, Du J. RNA splicing analysis contributes to reclassifying variants of uncertain significance and improves the diagnosis of monogenic disorders. J Med Genet 2022; 59:1010-1016. [PMID: 35121647 DOI: 10.1136/jmedgenet-2021-108013] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2021] [Accepted: 01/06/2022] [Indexed: 11/04/2022]
Abstract
BACKGROUND Numerous variants of uncertain significance (VUSs) have been identified by whole exome sequencing in clinical practice. However, VUSs are not currently considered medically actionable. OBJECTIVE To assess the splicing patterns of 49 VUSs in 48 families identified clinically to improve genetic counselling and family planning. METHODS Forty-nine participants with 49 VUSs were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Bioinformatic analysis was performed to preliminarily predict the splicing effects of these VUSs. RT-PCR and minigene analysis were used to assess the splicing patterns of the VUSs. According to the results obtained, couples opted for different methods of reproductive interventions to conceive a child, including prenatal diagnosis and preimplantation genetic testing (PGT). RESULTS Eleven variants were found to alter pre-mRNA splicing and one variant caused nonsense-mediated mRNA decay, which resulted in the reclassification of these VUSs as likely pathogenic. One couple chose to undergo in vitro fertilisation with PGT treatment; a healthy embryo was transferred and the pregnancy is ongoing. Three couples opted for natural pregnancy with prenatal diagnosis. One couple terminated the pregnancy because the fetus was affected by short-rib thoracic dysplasia and harboured the related variant. The infants of the other two couples were born and were healthy at their last recorded follow-up. CONCLUSION RNA splicing analysis is an important method to assess the impact of sequence variants on splicing in clinical practice and can contribute to the reclassification of a significant proportion of VUSs. RNA splicing analysis should be considered for genetic disease diagnostics.
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Affiliation(s)
- Wen-Bin He
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China.,National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China.,Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China
| | - Wen-Juan Xiao
- National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China.,Hunan Guangxiu Hospital, Medical College of Hunan Normal University, Changsha, Hunan, China
| | - Cong-Ling Dai
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China.,National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China.,Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China
| | - Yu-Rong Wang
- Hunan Guangxiu Hospital, Medical College of Hunan Normal University, Changsha, Hunan, China
| | - Xiu-Rong Li
- Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China
| | - Fei Gong
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China.,National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China.,Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China
| | - Lan-Lan Meng
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China.,Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China
| | - Chen Tan
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China
| | - Si-Cong Zeng
- Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Hunan Guangxiu Hospital, Medical College of Hunan Normal University, Changsha, Hunan, China
| | - Guang-Xiu Lu
- National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China.,Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China.,Hunan Guangxiu Hospital, Medical College of Hunan Normal University, Changsha, Hunan, China
| | - Ge Lin
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China.,National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China.,Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China
| | - Yue-Qiu Tan
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China.,National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China.,Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China.,Hunan Guangxiu Hospital, Medical College of Hunan Normal University, Changsha, Hunan, China
| | - Hao Hu
- Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China .,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China
| | - Juan Du
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China .,National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China.,Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.,Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China
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48
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Abstract
RNA-based therapeutics have entered the mainstream with seemingly limitless possibilities to treat all categories of neurological disease. Here, common RNA-based drug modalities such as antisense oligonucleotides, small interfering RNAs, RNA aptamers, RNA-based vaccines and mRNA drugs are reviewed highlighting their current and potential applications. Rapid progress has been made across rare genetic diseases and neurodegenerative disorders, but safe and effective delivery to the brain remains a significant challenge for many applications. The advent of individualized RNA-based therapies for ultra-rare diseases is discussed against the backdrop of the emergence of this field into more common conditions such as Alzheimer's disease and ischaemic stroke. There remains significant untapped potential in the use of RNA-based therapeutics for behavioural disorders and tumours of the central nervous system; coupled with the accelerated development expected over the next decade, the true potential of RNA-based therapeutics to transform the therapeutic landscape in neurology remains to be uncovered.
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Affiliation(s)
- Karen Anthony
- Centre for Physical Activity and Life Sciences, University of Northampton, Northampton, UK
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49
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Liu L, Yu C, Yan G. Identification of a novel heterozygous TSC2 splicing variant in a patient with Tuberous sclerosis complex: A case report. Medicine (Baltimore) 2022; 101:e28666. [PMID: 35060563 PMCID: PMC8772658 DOI: 10.1097/md.0000000000028666] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Accepted: 01/05/2022] [Indexed: 01/05/2023] Open
Abstract
RATIONALE Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by facial angiofibromas, epilepsy, intellectual disability, and the development of hamartomas in several organs, including the heart, kidneys, brain, and lungs. Mutations in either TSC1 or TSC2 result in dysregulated mTOR activation, leading to the occurrence of TSC. PATIENT CONCERNS A 44-year-old man was hospitalized for acute lumbago and hematuria. DIAGNOSIS The patient presented with facial angiofibromas, epilepsy, fibrous plaques, periungual fibroma, renal angiomyolipomas (AML), pulmonary lymphangioleiomyomatosis (LAM), liver hamartomas, and osteosclerosis. A diagnosis of TSC was made based on clinical manifestations. INTERVENTIONS Next-generation sequencing (NGS) was performed to screen for potential variants, which were verified using Sanger sequencing. The final variant was analyzed using a minigene assay. OUTCOMES A potentially pathogenic novel TSC2 variant (NM_000548.4, c.336_336 + 15delGGTAAGGCCCAGGGCG) was identified using NGS and confirmed using Sanger sequencing. The in vitro minigene assay showed that the variant c.336_336 + 15delGGTAAGGCCCAGGGCG caused erroneous integration of a 74 bp sequence into intron 4. This novel variant was not found in his unaffected parents or 100 unrelated healthy controls. LESSONS We identified a novel heterozygous TSC2 variant, c.336_336 + 15delGGTAAGGCCCAGGGCG, in a patient with classical TSC and demonstrated that this variant leads to aberrant splicing using a minigene assay. Our results extend the understanding of the mutational spectrum of TSC2.
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Affiliation(s)
- Linli Liu
- Department of Dermatology, Suining Central Hospital, Suining, People's Republic of China
| | - Chunshui Yu
- Department of Dermatology, Suining Central Hospital, Suining, People's Republic of China
| | - Gaowu Yan
- Department of Radiography, Suining Central Hospital, Suining, People's Republic of China
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50
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Lai HC, Ho UY, James A, De Souza P, Roberts TL. RNA metabolism and links to inflammatory regulation and disease. Cell Mol Life Sci 2021; 79:21. [PMID: 34971439 PMCID: PMC11072290 DOI: 10.1007/s00018-021-04073-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2021] [Revised: 09/29/2021] [Accepted: 10/22/2021] [Indexed: 11/29/2022]
Abstract
Inflammation is vital to protect the host against foreign organism invasion and cellular damage. It requires tight and concise gene expression for regulation of pro- and anti-inflammatory gene expression in immune cells. Dysregulated immune responses caused by gene mutations and errors in post-transcriptional regulation can lead to chronic inflammatory diseases and cancer. The mechanisms underlying post-transcriptional gene expression regulation include mRNA splicing, mRNA export, mRNA localisation, mRNA stability, RNA/protein interaction, and post-translational events such as protein stability and modification. The majority of studies to date have focused on transcriptional control pathways. However, post-transcriptional regulation of mRNA in eukaryotes is equally important and related information is lacking. In this review, we will focus on the mechanisms involved in the pre-mRNA splicing events, mRNA surveillance, RNA degradation pathways, disorders or symptoms caused by mutations or errors in post-transcriptional regulation during innate immunity especially toll-like receptor mediated pathways.
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Affiliation(s)
- Hui-Chi Lai
- Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia.
- South West Sydney Clinical School, UNSW Australia, Liverpool, NSW, Australia.
| | - Uda Y Ho
- School of Biomedical Sciences, Faculty of Medicine, University of Queensland, Brisbane, Australia
| | - Alexander James
- Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
| | - Paul De Souza
- Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
- School of Medicine, University of Wollongong, Wollongong, NSW, Australia
- School of Medicine, Western Sydney University, Macarthur, NSW, Australia
| | - Tara L Roberts
- Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
- South West Sydney Clinical School, UNSW Australia, Liverpool, NSW, Australia
- School of Medicine, Western Sydney University, Macarthur, NSW, Australia
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