Disease ‐causing variants of the conserved +2T of 5′ splice sites can be rescued by engineered U1snRNAs

The ability of variants of the spliceosomal U1snRNA to rescue splicing has been proven in several human disease models, but not for nucleotide changes at the conserved GT nucleotide of 5 ′ splice sites (5′ss), frequent and associated with severe phenotypes. Through minigene expression of variants at the 5′ss ofF9 intron 3 and splicing modulation by engineered U1snRNAs, we demonstrated that some nucleotide changes at the 5 ′ss +2T nucleotide can be rescued, thus expanding the applicability of this U1snRNA‐based approach. AbstractThe ability of variants of the spliceosomal U1snRNA to rescue splicing has been proven in several human disease models, but not for nucleotide changes at the conserved GT nucleotide of 5 ′ splice sites (5′ss), frequent and associated with severe phenotypes. Here, we focused on variants at the 5′ss of F9 intron 3, leading to factor IX (FIX) deficiency (hemophilia B). Through minigene expression, we demonstrated that all changes induce complete exon 3 skipping, which explains th e associated hemophilia B phenotype. Interestingly, engineered U1snRNAs remarkably increased the proportion of correct transcripts in the presence of the c.277+4A>G ( ∼60%) and also c.277+2T>C mutation ( ∼20%). Expression of splicing‐competent cDNA constructs indicated that the splicing rescue produces an appreciable increase of secreted FIX protein levels. These data provide the first experimental evidence that even part of variants at the conserved 5...

Source: Human Mutation - November 19, 2018 Category: Genetics & Stem Cells Authors: Daniela Scalet, Iva Maestri, Alessio Branchini, Francesco Bernardi, Mirko Pinotti, Dario Balestra Tags: BRIEF REPORT Source Type: research