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

ABSTRACT Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes ( EMGs) to determine the RNA and protein products generated by splice site variants ( n = 10) implicated in cystic fibrosis ( CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney ( HEK293) and CF bronchial epithelial ( CFBE41o-) cells. Quantification of variant induced aberrant m RNA 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 experimental 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 ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools. [ABSTRACT FROM AUTHOR]