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Leveraging splice‐affecting variant predictors and a minigene validation system to identify Mendelian disease‐causing variants among exon‐captured variants of uncertain significance |
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| Authors: | Zachry T. Soens Justin Branch Shijing Wu Zhisheng Yuan Yumei Li Hui Li Keqing Wang Mingchu Xu Lavan Rajan Fabiana L. Motta Renata T. Simões Irma Lopez‐Solache Radwan Ajlan David G. Birch Peiquan Zhao Fernanda B. Porto Juliana Sallum Robert K. Koenekoop Ruifang Sui Rui Chen |
| Affiliation: | 1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas;2. Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas;3. Department of Ophthalmology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China;4. Department of Ophthalmology and Visual Sciences, Paulista School of Medicine, Federal University of S?o Paulo, S?o Paulo, Brazil;5. Department of Retina and Vitreous, Ophthalmologic Center of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil;6. Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte Hospital – IEP/SCBH, Belo Horizonte, Minas Gerais, Brazil;7. McGill Ocular Genetics Laboratory and Centre, Department of Paediatric Surgery, Human Genetics, and Ophthalmology, McGill University Health Centre, Montreal, Quebec, Canada;8. Retina Foundation of the Southwest and Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas;9. Department of Ophthalmology, Xin Hua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China;10. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas;11. Department of Structural and Computational Biology & Molecular Biophysics, Baylor College of Medicine, Houston, Texas |
| Abstract: | The genetic heterogeneity of Mendelian disorders results in a significant proportion of patients that are unable to be assigned a confident molecular diagnosis after conventional exon sequencing and variant interpretation. Here, we evaluated how many patients with an inherited retinal disease (IRD) have variants of uncertain significance (VUS) that are disrupting splicing in a known IRD gene by means other than affecting the canonical dinucleotide splice site. Three in silico splice‐affecting variant predictors were leveraged to annotate and prioritize variants for splicing functional validation. An in vitro minigene system was used to assay each variant's effect on splicing. Starting with 745 IRD patients lacking a confident molecular diagnosis, we validated 23 VUS as splicing variants that likely explain disease in 26 patients. Using our results, we optimized in silico score cutoffs to guide future variant interpretation. Variants that alter base pairs other than the canonical GT‐AG dinucleotide are often not considered for their potential effect on RNA splicing but in silico tools and a minigene system can be utilized for the prioritization and validation of such splice‐disrupting variants. These variants can be overlooked causes of human disease but can be identified using conventional exon sequencing with proper interpretation guidelines. |
| Keywords: | inherited retinal degenerations minigene Mendelian disease molecular diagnosis noncanonical splicing variants variants of uncertain significance (VUS) |