This transition reflects the need to understand human variation on splicing and its effect on human diseases, which is of most interest for medical geneticists.
PCR and sequence analysis of whole blood RNA samples from population controls was then carried out for the 8 most promising variants to determine the effect of genetic variation on splicing of target genes.
mRNA analysis was useful to demonstrate the effect of two intronic variations on RNA splicing.
Comparisons of methods for missense variant tolerance, protein stability changes due to amino acid substitutions, and effects of variations on mRNA splicing are presented.
The pTB NdeI plasmid (generously provided by F. Pagani) is a strong support to examine the impact of intronic or/and exonic variations on aberrant splicing in transfected mammalian cells.
In addition to self-splicing to ligate two exons that are juxtaposed to a group I intron, several variations on self-splicing have been identified, including trans-splicing, which is used to correct gene sequences with mutations, a promising technique with potential therapeutic application [ 3, 4].
In the fourth dataset were negative controls, altogether 85 variations without effect on splicing.
Despite the early identifications of two associated intronic variations, their impacts on splicing have never been assessed yet neither independently nor in association.
SIFT, PolyPhen-2, MutationTaster, and Alamut (Interactive Biosoftware, Rouen, France) predicted the consequences of an identified amino acid substitution on protein structure/function and pathogenic potential, and rapidly assessed nucleotide and amino acid conservation, potential protein domain involvement, and nucleotide variation impact on splice site.
