Premium
Disease‐causing variants of the conserved +2T of 5′ splice sites can be rescued by engineered U1snRNAs
Author(s) -
Scalet Daniela,
Maestri Iva,
Branchini Alessio,
Bernardi Francesco,
Pinotti Mirko,
Balestra Dario
Publication year - 2019
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.23680
Subject(s) - minigene , biology , rna splicing , intron , exon , genetics , splice , alternative splicing , phenotype , mutation , nucleotide , gene , rna
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. 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 the 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′ss +2T nucleotide can be rescued, thus expanding the applicability of this U1snRNA‐based approach.