Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

d Assessment of the functional consequences of variants near splice sites isa major challenge in the 'agnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) plicated in cystic fibrosis (CF). Experimental results ere compared with the splicing predictions of eight in lwo tools. EMGs containing the full-length Cystic Fiosis Transmembrane Conductance Regulator (CFTR.) coding sequence and flanking intron sequences generated '1d-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE410-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585 1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with xperimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing Predicted 11/16 (HSF and ASSEI)A) and 10/16 (FsPlice and SplkePort) experimentally observed mRTsIA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in ilico tools. (C) 2014 wiley periodicals, Inc.