title: 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 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 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.
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Neeraj
authors: N
authors: Sharma
authors: Patrick R
authors: PR
authors: Sosnay
authors: Anabela S
authors: AS
authors: Ramalho
authors: Christopher
authors: C
authors: Douville
authors: Arianna
authors: A
authors: Franca
authors: Laura B
authors: LB
authors: Gottschalk
authors: Jeenah
authors: J
authors: Park
authors: Melissa
authors: M
authors: Lee
authors: Briana
authors: B
authors: Vecchio-Pagan
authors: Karen S
authors: KS
authors: Raraigh
authors: Margarida D
authors: MD
authors: Amaral
authors: Rachel
authors: R
authors: Karchin
authors: Garry R
authors: GR
authors: Cutting
mesh_terms: D002460:Cell Line; D003198:Computer Simulation; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D005821:Genetic Techniques; D006801:Humans; D009154:Mutation; D059368:RNA Isoforms / Q000032:analysis / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing
chemical_list: C505032:CFTR protein, human; D059368:RNA Isoforms; D022821:RNA Splice Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: CFTR; expression minigene; in silico tools; splicing