Evaluation of damaging effects of splicing mutations: Validation of an in vitro method for diagnostic laboratories

Background: Pre-mRNA splicing defects may have an important impact on clinical phenotype in several diseases, but often their pathogenic role is difficult to demonstrate. The aim of this study was to validate an in vitro method to assess the effects of putative splicing variants. Materials and methods: We studied three novel variants in vitro using a novel minigene approach and compared results with in silica and ex vivo strategies from patient samples. Results: For the c.1146C>T variant in the LMNA gene, in vitro and ex vivo studies were concordant with the prediction obtained by in silica tools, confirming the loss of 13 bp at the end of exon 6. In the second case (c.1140+1G>A, SCN5A gene), in vitro experiments identified the insertion of 94 intronic bp in exon 9 as well as exon 9 skipping, but these results were not correctly predicted by ex vivo data and in silica tools. In the third case (c.1608+1C>T, LMNA gene) in vitro and ex vivo studies suggested the recognition of an exonic cryptic site leading to the loss of 29 bp in exon 9, not predicted by in silica analysis. Conclusion: Our results revealed how in silica tools are often unreliable requiring "wet" RNA analysis. Since ex vivo studies are not always feasible, the use of an in vitro construct represents an efficient and useful method for the evaluation of damaging effects of unknown splicing variants, especially in diagnostic laboratories. (C) 2014 Elsevier B.V. All rights reserved.