Leveraging splice-affecting variant predictors and a minigene validation system to identify Mendelian disease-causing variants among exon-captured variants of uncertain significance

5 years ago

Leveraging splice-affecting variant predictors and a minigene validation system to identify Mendelian disease-causing variants among exon-captured variants of uncertain significance

Ruifang Sui, Hui Li, Lavan Rajan, Mingchu Xu, Keqing Wang, Peiquan Zhao, Shijing Wu, Irma Lopez-Solache, Radwan Ajlan, Renata T. Simões, Fabiana L. Motta, David G. Birch, Justin Branch, Robert K. Koenekoop, Yumei Li, Zhisheng Yuan, Rui Chen, Fernanda B. Porto, Juliana Sallum, Zachry T. Soens

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. Retinal disease patients were sequenced and screened following the flowchart for potential splicing variants with an uncertain functional impact. The effect on splicing caused by each variant was assessed using a minigene validation system.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/humu.23294