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Hum Mutat. 2014 Oct;35(10):1249-59. doi: 10.1002/humu.22624. Epub 2014 Sep 10.

Experimental assessment of splicing variants using expression minigenes and comparison with in silico predictions.

Author information

1
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.

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.

KEYWORDS:

CFTR; expression minigene; in silico tools; splicing

PMID:
25066652
PMCID:
PMC4425124
DOI:
10.1002/humu.22624
[Indexed for MEDLINE]
Free PMC Article

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