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

Neeraj Sharma, Patrick R. Sosnay, Anabela S. Ramalho, Christopher Douville, Arianna Franca, Laura B. Gottschalk, Jeenah Park, Melissa Lee, Briana Vecchio-Pagan, Karen S. Raraigh, Margarida D. Amaral, Rachel Karchin, Garry R. Cutting

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)1249-1259
Number of pages11
JournalHuman mutation
Volume35
Issue number10
DOIs
StatePublished - Oct 1 2014

Keywords

  • CFTR
  • Expression minigene
  • In silico tools
  • Splicing

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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