title: Evaluation of damaging effects of splicing mutations: validation of an in vitro method for diagnostic laboratories.
abstract: 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.
  
  
  METHODS
  We studied three novel variants in vitro using a novel minigene approach and compared results with in silico 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 silico 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 silico 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 silico analysis.
  
  
  CONCLUSIONS
  Our results revealed how in silico 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.
authors.affiliation: Vita-Salute San Raffaele University, Milan, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Di Resta
authors.affiliation: Vita-Salute San Raffaele University, Milan, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Manzoni
authors.affiliation: Department of Cardiology, Padre A. Micone Hospital, ASL 3 "Genovese," Genoa, Italy.
authors.forename: Massimo Zoni
authors.initials: MZ
authors.lastname: Berisso
authors.affiliation: Neurological Clinics, Department of Clinical and Experimental Medicine, University of Pisa, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Siciliano
authors.affiliation: Clinical Molecular Biology and Cytogenetics Laboratory, IRCCS San Raffaele Hospital, Milan, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Benedetti
authors.affiliation: Vita-Salute San Raffaele University, Milan, Italy; Clinical Molecular Biology and Cytogenetics Laboratory, IRCCS San Raffaele Hospital, Milan, Italy. Electronic address: ferrari.maurizio@hsr.it.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
mesh_terms: D001483:Base Sequence; D019411:Clinical Laboratory Techniques / Q000379:methods*; D003198:Computer Simulation; D005091:Exons / Q000235:genetics; D006801:Humans; D034904:Lamin Type A / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009154:Mutation; D012326:RNA Splicing / Q000235:genetics*; D055815:Young Adult
chemical_list: C494847:LMNA protein, human; D034904:Lamin Type A
keywords: Expression vectors; In silico tools; In vitro studies; Minigene; RNA analysis; Splicing defects