RNA variant assessment using transactivation and transdifferentiation,American Journal of Human Genetics

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RNA variant assessment using transactivation and transdifferentiation
American Journal of Human Genetics ( IF 8.1 ) Pub Date : 2024-07-30 , DOI: 10.1016/j.ajhg.2024.06.018
Emmylou C Nicolas-Martinez ₁ , Olivia Robinson ₁ , Christian Pflueger ₂ , Alison Gardner ₃ , Mark A Corbett ₄ , Tarin Ritchie ₃ , Thessa Kroes ₃ , Clare L van Eyk ₄ , Ingrid E Scheffer ₅ , Michael S Hildebrand ₆ , Jean-Vianney Barnier ₇ , Véronique Rousseau ₇ , David Genevieve ₈ , Virginie Haushalter ₉ , Amélie Piton ₉ , Anne-Sophie Denommé-Pichon ₁₀ , Ange-Line Bruel ₁₀ , Sophie Nambot ₁₀ , Bertrand Isidor ₁₀ , John Grigg ₁₁ , Tina Gonzalez ₁₂ , Sondhya Ghedia ₁₂ , Rhett G Marchant ₁₃ , Adam Bournazos ₁₄ , Wui-Kwan Wong ₁₅ , Richard I Webster ₁₆ , Frances J Evesson ₁₇ , Kristi J Jones ₁₈ , , Sandra T Cooper ₁₇ , Ryan Lister ₁₉ , Jozef Gecz ₂₀ , Lachlan A Jolly ₁

Affiliation

The Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia; School of Biomedicine, University of Adelaide, Adelaide, SA 5005, Australia.
Harry Perkins Institute of Medical Research, Nedlands, WA 6009, Australia; Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Crawley, WA 6009, Australia; The Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia.
The Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia; Adelaide Medical School, University of Adelaide, Adelaide, SA 5005, Australia.
The Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia; Adelaide Medical School, University of Adelaide, Adelaide, SA 5005, Australia; The Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia.
Epilepsy Research Centre, Department of Medicine, The University of Melbourne, Austin Health, Heidelberg, VIC 3084, Australia; Murdoch Children's Research Institute, Parkville, VIC 3052, Australia; Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, VIC 3052, Australia.
Epilepsy Research Centre, Department of Medicine, The University of Melbourne, Austin Health, Heidelberg, VIC 3084, Australia; Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, VIC 3052, Australia; The Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia.
Institut des Neurosciences Paris-Saclay, UMR 9197, CNRS, Université Paris-Saclay, Saclay, France.
Montpellier University, Inserm U1183, Reference Center for Rare Diseases Developmental Anomaly and Malformative Syndromes, Genetics Department, Montpellier Hospital, Montpellier, France.
Genetic Diagnosis Laboratory, Strasbourg University Hospital, Strasbourg, France.
CRMRs "Anomalies du Développement et syndromes malformatifs" et "Déficiences Intellectuelles de causes rares", Centre de Génétique, CHU Dijon, Dijon, France; INSERM UMR1231, GAD "Génétique des Anomalies du Développement," FHU-TRANSLAD, University of Burgundy, Dijon, France.
Speciality of Ophthalmology, Save Sight Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2000, Australia.
Department of Clinical Genetics, Royal North Shore Hospital, St Leonards, NSW 2065, Australia.
Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Westmead, NSW 2145, Australia; Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2000, Australia.
Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Westmead, NSW 2145, Australia; Children's Medical Research Institute, Westmead, NSW 2145, Australia.
Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Westmead, NSW 2145, Australia; Children's Medical Research Institute, Westmead, NSW 2145, Australia; Department of Paediatric Neurology, Children's Hospital at Westmead, Sydney, NSW 2000, Australia.
Department of Paediatric Neurology, Children's Hospital at Westmead, Sydney, NSW 2000, Australia.
Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Westmead, NSW 2145, Australia; Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2000, Australia; Children's Medical Research Institute, Westmead, NSW 2145, Australia.
Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Westmead, NSW 2145, Australia; Children's Medical Research Institute, Westmead, NSW 2145, Australia; Department of Clinical Genetics, Children's Hospital at Westmead, Sydney, NSW 2000, Australia.
Harry Perkins Institute of Medical Research, Nedlands, WA 6009, Australia; Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Crawley, WA 6009, Australia.
The Robinson Research Institute, University of Adelaide, Adelaide, SA 5005, Australia; Adelaide Medical School, University of Adelaide, Adelaide, SA 5005, Australia; South Australian Health and Medical Research Institute, Adelaide, SA 5000, Australia.

Understanding the impact of splicing and nonsense variants on RNA is crucial for the resolution of variant classification as well as their suitability for precision medicine interventions. This is primarily enabled through RNA studies involving transcriptomics followed by targeted assays using RNA isolated from clinically accessible tissues (CATs) such as blood or skin of affected individuals. Insufficient disease gene expression in CATs does however pose a major barrier to RNA based investigations, which we show is relevant to 1,436 Mendelian disease genes. We term these “silent” Mendelian genes (SMGs), the largest portion (36%) of which are associated with neurological disorders. We developed two approaches to induce SMG expression in human dermal fibroblasts (HDFs) to overcome this limitation, including CRISPR-activation-based gene transactivation and fibroblast-to-neuron transdifferentiation. Initial transactivation screens involving 40 SMGs stimulated our development of a highly multiplexed transactivation system culminating in the 6- to 90,000-fold induction of expression of 20/20 (100%) SMGs tested in HDFs. Transdifferentiation of HDFs directly to neurons led to expression of 193/516 (37.4%) of SMGs implicated in neurological disease. The magnitude and isoform diversity of SMG expression following either transactivation or transdifferentiation was comparable to clinically relevant tissues. We apply transdifferentiation and/or gene transactivation combined with short- and long-read RNA sequencing to investigate the impact that variants in , , , and have on RNA using HDFs derived from affected individuals. Transactivation and transdifferentiation represent rapid, scalable functional genomic solutions to investigate variants impacting SMGs in the patient cell and genomic context.

中文翻译：

使用反式激活和转分化评估 RNA 变异

了解剪接和无义变异对 RNA 的影响对于解决变异分类及其对精准医学干预的适用性至关重要。这主要是通过涉及转录组学的 RNA 研究，然后使用从受影响个体的血液或皮肤等临床可及组织 (CAT) 中分离的 RNA 进行靶向测定来实现的。然而，CAT 中疾病基因表达不足确实对基于 RNA 的研究构成了主要障碍，我们证明这与 1,436 个孟德尔疾病基因相关。我们将这些“沉默”孟德尔基因 (SMG) 称为“沉默”孟德尔基因，其中最大部分 (36%) 与神经系统疾病相关。我们开发了两种在人真皮成纤维细胞 (HDF) 中诱导 SMG 表达的方法来克服这一限制，包括基于 CRISPR 激活的基因反式激活和成纤维细胞到神经元的转分化。涉及 40 个 SMG 的初始反式激活筛选刺激了我们开发高度多重反式激活系统，最终在 HDF 中测试了 20/20 (100%) SMG 的表达诱导 6 至 90,000 倍。 HDF 直接转分化为神经元导致 193/516 (37.4%) 的 SMG 表达，与神经系统疾病有关。反式激活或转分化后 SMG 表达的幅度和亚型多样性与临床相关组织相当。我们将转分化和/或基因反式激活与短读长和长读长 RNA 测序相结合，利用来自受影响个体的 HDF 来研究、、和中的变异对 RNA 的影响。反式激活和转分化代表了快速、可扩展的功能基因组解决方案，用于研究影响患者细胞和基因组环境中 SMG 的变异。

更新日期：2024-07-30

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